Pressure Manifold and Culture Module

ABSTRACT

A culture module is contemplated that allows the perfusion and optionally mechanical actuation of one or more microfluidic devices, such as organ-on-a-chip microfluidic devices comprising cells that mimic at least one function of an organ in the body.

FIELD OF THE INVENTION

A perfusion manifold assembly is contemplated that allows for perfusionof a microfluidic device, such as an organ on a chip microfluidic devicecomprising cells that mimic cells in an organ in the body or at leastone function of an organ, that is detachably linked with said assemblyso that fluid enters ports of the microfluidic device from a fluidreservoir, optionally without tubing, at a controllable flow rate. Adrop-to-drop connection scheme is contemplated as one embodiment forputting a microfluidic device in fluidic communication with a fluidsource or another microfluidic device, including but not limited to,putting a microfluidic device in fluidic communication with theperfusion manifold assembly.

BACKGROUND OF THE INVENTION

Two-dimensional (2D) monolayer cell culture systems have been used formany years in biological research. The most common cell culture platformis the two-dimensional (2D) monolayer cell culture in petri dishes orflasks. Although such 2D in vitro models are less expensive than animalmodels and are conducive to systematic, and reproducible quantitativestudies of cell physiology (e.g., in drug discovery and development),the physiological relevance of the information retrieved from in vitrostudies to in vivo system is often questionable. It has now been widelyaccepted that three-dimensional (3D) cell culture matrix promotes manybiological relevant functions not observed in 2D monolayer cell culture.Said another way, 2D cell culture systems do not accurately recapitulatethe structure, function, physiology of living tissues in vivo.

U.S. Pat. No. 8,647,861 describes microfluidic “organ-on-chip” devicescomprising living cells on membranes in microchannels exposed to culturefluid at a flow rate. In contrast to static 2D culture, microchannelsallow the perfusion of cell culture medium throughout the cell cultureduring in vitro studies and as such offer a more in vivo-like physicalenvironment. In simple terms, an inlet port allows injection of cellculture medium into a cell-laden microfluidic channel or chamber, thusdelivering nutrients and oxygen to cells. An outlet port then permitsthe exit of remaining medium as well as harmful metabolic by-products.

While such microfluidic devices are an improvement over traditionalstatic tissue culture models, the small size, scale and interface ofthese devices makes fluid handling difficult. What is needed is a way tocontrol perfusion of these devices in a manner whereby fluid pressurecreates a flow rate that applies a desired fluid shear stress to theliving cells. Ideally, the solution should provide for a simple userworkflow.

SUMMARY OF THE INVENTION

The present invention contemplates a number of devices separately and incombination. The present invention contemplates a perfusion manifoldassembly (also referred to as a cartridge, pod or perfusion disposable,whether or not there is any requirement or intent to dispose of thecomponent) is contemplated that retains one or more microfluidicdevices, such as “organ-on-a-chip” microfluidic devices (or simply“microfluidic chip”) that comprise cells that mimic at least onefunction of an organ in the body, and allow the perfusion and optionallythe mechanical actuation of said microfluidic devices, optionallywithout tubing. The present invention contemplates a number ofembodiments of the perfusion manifold assembly. However, it is notintended that the present invention be limited to these embodiments. Forexample, the present invention contemplates combining features fromdifferent embodiments (as discussed below). In addition, the presentinvention contemplates removing features from the embodiments (asdiscussed below). Furthermore, the present invention contemplatessubstituting features in the embodiments (as discussed below).

A culture module is contemplated that allows the perfusion andoptionally mechanical actuation of one or more microfluidic devices,such as organ-on-a-chip microfluidic devices comprising cells that mimicat least one function of an organ in the body. In one embodiment, themicrofluidic device comprises a top channel, a bottom channel, and amembrane separating at least a portion of said top and bottom channels.In one embodiment, the microfluidic device comprises cells on themembrane and/or in or on the channels. In one embodiment, the culturemodule comprises a pressure manifold that allows for perfusion of amicrofluidic device, such as an “organ on chip” microfluidic devicecomprising cells that mimic cells in an organ in the body or at leastone function of an organ, that is optionally retained in contact with aperfusion disposable and detachably linked with said assembly so thatfluid enters ports of the microfluidic device from a fluid reservoir,optionally without tubing, at a controllable flow rate. The perfusiondisposable can be used separately from the culture module, and themicrofluidic device or chip can be used separately from the perfusiondisposable. In one embodiment, the present invention contemplates a(moving or non-moving) pressure manifold configured to mate with one ormore microfluidic devices (such as any one of the perfusion manifoldassembly embodiments described herein) with integrated valves that canprevent gas leaks when not mated with a microfluidic device.

A drop-to-drop connection scheme is contemplated as one embodiment forputting a microfluidic device in fluidic communication with a fluidsource or another microfluidic device, including but not limited to,putting a microfluidic device in fluidic communication with a perfusiondisposable. In one embodiment, the microfluidic device comprises a topchannel, a bottom channel, and a membrane separating at least a portionof said top and bottom channels. In one embodiment, the microfluidicdevice comprises cells on the membrane and/or in or on the channels.

A pressure lid is contemplated that allows for the pressurization of oneor more reservoirs within a perfusion disposable or perfusion manifoldassembly (or other microfluidic device), the pressure lid being movableor removably attached to said perfusion disposable or other microfluidicdevice to allow improved access to elements (e.g. reservoirs) within.The pressure lid can be removed from the perfusion disposable and theperfusion disposable can be used without the lid. In one embodiment, theperfusion disposable comprises a microfluidic chip, and the chipcomprises a top channel, a bottom channel, and a membrane separating atleast a portion of said top and bottom channels. In one embodiment, themicrofluidic chip comprises cells on the membrane and/or in or on thechannels.

A method for pressure control is contemplated to allow the control offlow rate (while perfusing cells) despite limitations of common pressureregulators. Rather than having the pressure controllers (or actuators)of a culture module “on” all of the time (or at just one setpoint), inone embodiment, they are switched “on” and “off” (or between two or moresetpoints) in a pattern. Accordingly, the switching pattern may beselected such that the average value of pressure acting liquid in one ormore reservoirs of an engaged perfusion disposable (containing amicrofluidic device or chip) corresponds to a desired value. In oneembodiment, the microfluidic device comprises a top channel, a bottomchannel, and a membrane separating at least a portion of said top andbottom channels. In one embodiment, the microfluidic device comprisescells on the membrane and/or in or on the channels.

In one embodiment, the perfusion manifold assembly comprises i) a coveror lid configured to serve as the top of ii) one or more fluidreservoirs, iii) a capping layer under said fluid reservoir(s), iv) afluidic backplane under, and in fluidic communication with, said fluidreservoir(s), said fluidic backplane comprising a resistor, and v) aprojecting member or skirt (for engaging the microfluidic device or acarrier containing a microfluidic device). As noted above, the cover orlid can be removed and the perfusion manifold assembly can still beused. In one embodiment, the assembly further comprises fluid portspositioned at the bottom of the fluidic backplane. In one embodiment,the capping layer caps the fluid backplane. Without being bound bytheory of any particular mechanism, it is believed that these resistorsserve to stabilize the flow of fluid coming from the reservoirs so thata stable flow can be delivered to the microfluidic device, and/or theyserve to provide a means for translating reservoir pressure to perfusionflow rate. In one embodiment, the lid is held onto the reservoir using aradial seal. This does not require an applied pressure to create a seal.In another embodiment, the lid is held onto the reservoir using one ormore clips, screws or other retention mechanisms. In one embodiment, theprojecting member or skirt is engaged with a microfluidic chip. In oneembodiment, the microfluidic chip comprises a top channel, a bottomchannel, and a membrane separating at least a portion of said top andbottom channels. In one embodiment, the microfluidic device comprisescells on the membrane and/or in or on the channels.

In one embodiment, the perfusion manifold assembly comprises i) one ormore fluid reservoirs, and ii) a fluidic backplane under, and in fluidiccommunication with, said fluid reservoir(s), said fluidic backplanecomprising fluid channels that terminate a ports. In one embodiment, thefluidic backplane comprises a resistor. In one embodiment, the perfusionmanifold assembly further comprises iii) a projecting member or skirt.In one embodiment, the skirt comprises a guide mechanism (for engagingthe microfluidic device or a carrier containing a microfluidic device).In one embodiment, the guide mechanism comprises a guide shaft or ahole, groove, orifice or other cavity configured to accept a guideshaft. In one embodiment, the guide mechanism comprises (external orinternal) guide tracks. In one embodiment, the guide tracks are sidetracks (for engaging the microfluidic device or carrier). In oneembodiment, the perfusion manifold assembly may further include acapping layer that caps the fluidic backplane. The embodiment mayfurther optionally include a cover or lid. In one embodiment, the lid isheld onto the reservoir using a radial seal. This does not require anapplied pressure to create a seal. In another embodiment, the lid isheld onto the reservoir using one or more clips, screws or otherretention mechanisms. In one embodiment, fluidic ports are at the bottomof the fluidic backplane. In one embodiment, the projecting member orskirt is engaged with a microfluidic chip. In one embodiment, themicrofluidic chip comprises a top channel, a bottom channel, and amembrane separating at least a portion of said top and bottom channels.In one embodiment, the microfluidic device comprises cells on themembrane and/or in or on the channels.

In one embodiment, the perfusion manifold assembly comprises i) one ormore fluid reservoirs, ii) a fluidic backplane under, and in fluidiccommunication with, said fluid reservoir(s), said fluidic backplanecomprising a resistor, and iii) a projecting member or skirt (forengaging the microfluidic device or a cattier containing a microfluidicdevice). The embodiment may further include a capping layer that capsthe fluidic backplane. The embodiment may further optionally include acover or lid. In one embodiment, the lid is held onto the reservoirusing a radial seal. This does not require an applied pressure to createa seal. In another embodiment, the lid is held onto the reservoir usingone or more clips, screws or other retention mechanisms. In oneembodiment, fluidic ports are at the bottom of the fluidic backplane. Inone embodiment, the projecting member or skirt is engaged with amicrofluidic chip. In one embodiment, the microfluidic chip comprises atop channel, a bottom channel, and a membrane separating at least aportion of said top and bottom channels. In one embodiment, themicrofluidic device comprises cells on the membrane and/or in or on thechannels.

In one embodiment, the perfusion manifold assembly comprises i) one ormore fluid reservoirs, ii) a fluidic backplane under, and in fluidiccommunication with, said fluid reservoir(s), and iii) a capping layerthat caps the fluidic backplane. In one embodiment, said fluidicbackplane comprising one or more resistors. In one embodiment, theassembly further comprises optionally iv) a projecting member or skirt(for engaging the microfluidic device or a carrier containing themicrofluidic device). The embodiment may further optionally include acover or lid. In some embodiments, attachment of a microfluidic deviceto the perfusion disposable is through an engagement with the skirt.However, in other embodiments, attachment is achieved directly with theassembly (without the skirt or other outward extension). In oneembodiment, the projecting member or skirt is engaged with amicrofluidic chip. In one embodiment, the microfluidic chip comprises atop channel, a bottom channel, and a membrane separating at least aportion of said top and bottom channels. In one embodiment, themicrofluidic device comprises cells on the membrane and/or in or on thechannels.

In one embodiment, the present invention contemplates a perfusionmanifold assembly, comprising i) one or more fluid reservoirs, ii) afluidic backplane positioned under, and in fluidic communication with,said fluid reservoirs, said fluidic backplane comprising a fluidresistor and fluid channels that terminate at ports, and iii) aprojecting member or skirt having one or more side tracks. In oneembodiment, the ports are positioned at the bottom of the fluidicbackplane. In one embodiment, said one or more side tracks areconfigured for engaging a microfluidic device positioned in amicrofluidic device carrier having one or more outer edges configured toslidably engage said one or more side tracks. In one embodiment ofslidably engaging, the linking approach to the perfusion manifoldcomprises 1) a sliding action, 2) a pivoting movement, and 3) a snap fitso as to provide alignment and fluidic connection in a single action. Inthe 1) sliding step, the chip (or other microfluidic device) is in thecarrier, which slides along to align the fluidic ports. In the 2) pivotstep, the carrier and chip (or other microfluidic device) is pivoteduntil ports come into fluid contact. In the 3) clip or snap fit step,the force needed to provide a secure seal is provided. In oneembodiment, the projecting member or skirt is engaged with amicrofluidic chip. In one embodiment, the microfluidic chip comprises atop channel, a bottom channel, and a membrane separating at least aportion of said top and bottom channels. In one embodiment, themicrofluidic device comprises cells on the membrane and/or in or on thechannels.

In one embodiment, the carrier has a cutout or “window” (e.g. atransparent window) for imaging (e.g. with a microscope) the cellswithin the microfluidic chip. In one embodiment, there is acorresponding cutout or window (e.g. transparent) in the perfusiondisposable. In one embodiment, the microfluidic device comprisesfeatures of the carrier to avoid the need for a separate substrate. Inone embodiment, the microfluidic device comprises a top channel, abottom channel, and a membrane separating at least a portion of said topand bottom channels. In one embodiment, the microfluidic devicecomprises cells on the membrane and/or in or on the channels.

In one embodiment, the present invention contemplates a perfusionmanifold assembly, comprising i) one or more fluid reservoirs, ii) afluidic backplane positioned under, and in fluidic communication with,said fluid reservoirs, said fluidic backplane comprising a fluidresistor and fluid channels that terminate at iii) a projecting memberor skirt having one or more fluid ports and one or more side tracks. Inone embodiment, said one or more side tracks are configured for engaginga microfluidic device positioned in a microfluidic device carrier havingone or more outer edges configured to slidably engage said one or moreside tracks. In one embodiment of slidably engaging, the linkingapproach to the perfusion manifold comprises 1) a sliding action, 2) apivoting movement, and 3) a snap fit so as to provide alignment andfluidic connection in a single action. In the 1) sliding step, the chip(or other microfluidic device) is in the carrier, which slides along toalign the fluidic ports. In the 2) pivot step, the carrier and chip (orother microfluidic device) is pivoted until ports come into fluidcontact. In the 3) clip or snap fit step, the force needed to provide asecure seal is provided. In one embodiment, the microfluidic devicecomprises features of the carrier to avoid the need for a separatesubstrate. In one embodiment, the carrier has a cutout or “window” (e.g.a transparent window) for imaging (e.g. with a microscope). In oneembodiment, there is a corresponding cutout or window (e.g. transparent)in the perfusion disposable (e.g. in the fluid layer). In oneembodiment, the present invention contemplates control of the focalplane position and alignment (flatness vs. the microscope stage) atwhich the chip sits. It is preferred that the required working distancefor imaging be minimized (since larger working distances put more burdenon the objective). It is not intended that the present invention belimited by the imaging approach; imaging can be upright (objective fromabove) or inverted (objective from the bottom). While certainembodiments have a cutout or window on only one side for certain imagingmodalities (e.g. epifluorescence), in a preferred embodiment the presentinvention contemplates cutouts or windows on both sides of the chip toenable transmitted light imaging. In one embodiment, said resistorcomprises serpentine channels. In one embodiment, said fluidic backplaneis made of Cyclo Olefin Polymer (COP) (such as Zeonor 1420R, which iscommercially available) and comprises linear fluid channels in fluidiccommunication with said serpentine channels, said linear channelsterminating at one or more ports. In one embodiment, the skirt is madefrom polycarbonate (PC). In one embodiment, the assembly furthercomprising a cover for said fluid reservoirs, wherein said covercomprises a plurality of ports optionally associated with filters. Insome embodiments, the cover ports comprise through-holes and filterspositioned above corresponding holes in a gasket. In some embodiments,the cover comprises one or more channels that route one or more of theports (such that the port is not a simple through-hole). In oneembodiment, said side track comprises a closed first end proximal tosaid reservoirs and an opened second end distal to said reservoirs, saidopened end comprising an angled slide for engaging said one or moreouter edges of said microfluidic device carrier. In one embodiment, saidside track comprises a linear region between said closed first end andsaid opened second end. In one embodiment, the projecting member orskirt is engaged with a microfluidic chip. In one embodiment, themicrofluidic chip comprises a top channel, a bottom channel, and amembrane separating at least a portion of said top and bottom channels.In one embodiment, the microfluidic device comprises cells on themembrane and/or in or on the channels.

The present invention also contemplates systems comprising perfusionmanifold assemblies. In one embodiment, the present inventioncontemplates a system, comprising: a) a perfusion manifold assembly,comprising i) one or more fluid reservoirs, ii) a fluidic backplanepositioned under, and in fluidic communication with, said fluidreservoirs, and iii) a skirt or other projecting member; and b) amicrofluidic device or chip engaged with the perfusion manifold assemblythrough said skirt. In one embodiment, the microfluidic device isengaged in a detachable manner. In one embodiment, the microfluidicdevice is engaged in a manner that is not detachable (e.g. a one-timeconnection) whether through a locking mechanism or by using adhesives(e.g. an adhesive layer to assist with the quality of the fluidic seal).In one embodiment, said skirt has a guide mechanism for engaging saidmicrofluidic device. In one embodiment, the guide mechanism comprises aguide shaft or a hole, groove, orifice or other cavity configured toaccept a guide shaft. In one embodiment, said guide mechanism comprises(external or internal) guide tracks. In one embodiment, said guidetracks are side tracks. In one embodiment, said microfluidic device orchip is in a carrier and said carrier is engaged with the perfusionmanifold assembly through said side tracks of said skirt. In oneembodiment, the microfluidic device has one or more features of acarrier so as to avoid the need for an additional substrate such as acarrier. In one embodiment, the microfluidic device comprises a topchannel, a bottom channel, and a membrane separating at least a portionof said top and bottom channels. In one embodiment, the microfluidicdevice comprises cells on the membrane and/or in or on the channels. Inone embodiment, the assembly further comprising a cover or coverassembly for said fluid reservoirs, wherein said cover comprises aplurality of ports optionally associated with filters. In someembodiments, the cover ports comprise through-holes and filterspositioned above corresponding holes in a gasket. In some embodiments,the cover comprises one or more channels that route one or more of theports (such that the port is not a simple through-hole).

In one embodiment, the present invention contemplates a system,comprising: a) a perfusion manifold assembly, comprising i) one or morefluid reservoirs, ii) a fluidic backplane positioned under, and influidic communication with, said fluid reservoirs, said fluidicbackplane comprising a fluid resistor and fluid channels that terminateat fluid outlet ports at the bottom of said backplane, and iii) a skirtor other projecting member having one or more side tracks; and b) amicrofluidic device positioned in a carrier, said carrier having one ormore outer edges, said outer edges detachably engaging said one or moreside tracks of said skirt, said microfluidic device comprising i)microchannels in fluidic communication with said perfusion manifoldassembly via ii) one or more inlet ports on a iii) mating surface,wherein said one or more fluid inlet ports of said microfluidic deviceare positioned against said one or more fluid outlet ports of saidperfusion manifold assembly under conditions such that fluid flows fromsaid fluid reservoirs of said perfusion manifold assembly through saidone or more fluid outlet ports into said one or more fluid inlet portsof said microfluidic device. In one embodiment, the carrier is engagedin a detachable manner. In one embodiment, the carrier is engaged in amanner that is not detachable (e.g. a one-time connection) whetherthrough a locking mechanism or by using adhesives (e.g. an adhesivelayer to assist with the quality of the fluidic seal). In oneembodiment, the microfluidic device comprises a top channel, a bottomchannel, and a membrane separating at least a portion of said top andbottom channels. In one embodiment, the microfluidic device comprisescells on the membrane and/or in or on the channels. In one embodiment,the assembly further comprising a cover for said fluid reservoirs,wherein said cover comprises a plurality of openings associated withchannels. In one embodiment, the assembly further comprising a cover forsaid fluid reservoirs, wherein said cover comprises a plurality of portsoptionally associated with filters. In some embodiments, the cover portscomprise through-holes and filters positioned above corresponding holesin a gasket. In some embodiments, the cover comprises one or morechannels that route one or more of the ports (such that the port is nota simple through-hole).

In one embodiment, the present invention contemplates a system,comprising: a) a perfusion manifold assembly, comprising i) one or morefluid reservoirs, ii) a fluidic backplane positioned under, and influidic communication with, said fluid reservoirs, said fluidicbackplane comprising a fluid resistor and fluid channels that terminateat iii) a skirt having one or more fluid outlet ports and one or moreside tracks; and b) a microfluidic device positioned in a carrier, saidcarrier having one or more outer edges, said outer edges detachablyengaging said one or more side tracks of said skirt, said microfluidicdevice comprising i) microchannels in fluidic communication with saidperfusion manifold assembly via ii) one or more inlet ports on a iii)mating surface, wherein said one or more fluid inlet ports of saidmicrofluidic device are positioned against said one or more fluid outletports of said skirt of said perfusion manifold assembly under conditionssuch that fluid flows from said fluid reservoirs of said perfusionmanifold assembly through said one or more fluid outlet ports into saidone or more fluid inlet ports of said microfluidic device. In oneembodiment, the microfluidic device comprises a top channel, a bottomchannel, and a membrane separating at least a portion of said top andbottom channels. In one embodiment, the microfluidic device comprisescells on the membrane and/or in or on the channels. In a preferredembodiment, said microfluidic device comprises living cells perfusedwith fluid from said fluid reservoirs. In one embodiment, the assemblyfurther comprising a cover for said fluid reservoirs, wherein said covercomprises a plurality of ports optionally associated with filters. Insome embodiments, the cover ports comprise through-holes and filterspositioned above corresponding holes in a gasket. In some embodiments,the cover comprises one or more channels that route one or more of theports (such that the port is not a simple through-hole).

In a particularly preferred embodiment, said microfluidic device or chip(whether positioned in a carrier or not) comprises at least twodifferent cell types that function together in a manner that mimic oneor more functions of cells in an organ in the body. In one embodiment,the microfluidic device comprises a membrane having top and bottomsurfaces, said top surface comprising a first cell type, said bottomsurface comprises a second cell type. In one embodiment, themicrofluidic device comprises a top channel, a bottom channel, and amembrane separating at least a portion of said top and bottom channels.In one embodiment, said first cell type is epithelial cells and saidsecond cell type is endothelial cells. In a preferred embodiment, saidmembrane is porous (e.g. porous to fluid, gases, cytokines and othermolecules, and, in some embodiments, porous to cells, permitting cellsto transmigrate the membrane).

In one embodiment, the present invention contemplates a method ofseeding cells into a microfluidic chip (e.g. having ports associatedwith one or more microfluidic channels), the method comprising a)providing i) a chip at least partially contained in a carrier, ii)cells, iii) a seeding guide and iv) a stand with portions configured toaccept at least one seeding guide in a stable mounted position; b)engaging said seeding guide with said carrier to create an engagedseeding guide; c) mounting said engaged seeding guide on said stand, andd) seeding said cells into said chip while said seeding guide is in astable mounted position. In one embodiment, the seeding guide isconfigured (e.g. with guide tracks) to engage the edges of said carrier.In one embodiment, the seeding guide has side tracks (similar oridentical to those in the skirt of one embodiment of the perfusionmanifold assembly) to engage the edges of said carrier. In oneembodiment of this method, a plurality of seeding guides are mounted onthe stand, permitting a plurality of chips to be seeded with cells. Inone embodiment, the microfluidic chip comprises a top channel, a bottomchannel, and a membrane separating at least a portion of said top andbottom channels. In one embodiment, the microfluidic chip, after saidseeding, comprises cells on the membrane and/or in or on the channels.In one embodiment, the method further comprises, after said seeding ofstep d), the steps of e) disengaging said carrier from said seedingguide and f) engaging said perfusion manifold assembly with said carriercomprising said microfluidic chip comprising cells.

In one embodiment, the present invention contemplates a method ofseeding cells into a microfluidic chip (e.g. having ports associatedwith one or more microfluidic channels), the method comprising a)providing i) a chip at least partially contained in a seeding guide, ii)cells and iii) a stand with portions configured to accept at least oneseeding guide in a stable mounted position; b) engaging said stand withsaid seeding guide; and c) seeding said cells into said chip while saidseeding guide is in a stable mounted position. In one embodiment of thismethod, a plurality of seeding guides is engaged with said stand,permitting a plurality of chips to be seeded with cells. In oneembodiment of this method, there is no chip carrier. In anotherembodiment, the chip carrier serves as the seeding guide (without aseparate seeding guide structure engaging the carrier).

In a preferred embodiment, said carrier further comprises a lockingmechanism for restricting movement of the carrier when said one or morefluid inlet ports of said microfluidic device are positioned againstsaid one or more fluid outlet ports of said perfusion manifold assembly.It is not intended that the present invention be limited to the natureof the locking mechanism. In one embodiment, the locking mechanism isselected from the group consisting of a clip, a clamp, a stud, and ascrew. In one embodiment, the locking mechanism engages in a frictionfit. The locking mechanism can permit either detachable engagement orengagement that is not detachable.

The present invention also contemplates methods of perfusing cellsutilizing a perfusion manifold assembly. In one embodiment, the presentinvention contemplates a method of perfusing cells, comprising: A)providing a) a perfusion manifold assembly comprising i) one or morefluid reservoirs, ii) a fluidic backplane positioned under, and influidic communication with, said fluid reservoirs, said fluidicbackplane comprising fluid channels that terminate at outlet ports, andiii) a skirt or other projecting member comprising a guide mechanism;and b) a microfluidic device positioned in a carrier, said carrierconfigured to engage said guide mechanism of said skirt, saidmicrofluidic device comprising i) living cells, and ii) microchannels influidic communication with ii) one or more inlet ports on a iii) matingsurface; B) positioning said carrier such that engages of said guidemechanism of said skirt; and C) moving said carrier until said one ormore fluid inlet ports of said microfluidic device are positionedagainst said one or more fluid outlet ports of said perfusion manifoldassembly under conditions such that said microfluidic device is linkedand fluid flows from said fluid reservoirs of said perfusion manifoldassembly through said one or more fluid outlet ports into said one ormore fluid inlet ports and into said microchannels of said microfluidicdevice, thereby perfusing said cells. In one embodiment, the fluidicbackplane comprises a fluid resistor. In one embodiment, the guidemechanism comprises a guide shaft or a hole, groove, orifice or othercavity configured to accept a guide shaft. In one embodiment, the guidemechanism comprises (external or internal) guide tracks. In oneembodiment, said guide tracks are side tracks. In one embodiment, saidcarrier comprises one or more outer edges, said outer edges configuredfor engaging said one or more side tracks of said skirt. In oneembodiment, the moving of step C) comprises sliding said carrier alongsaid side tracks until said inlet and outlet ports are positionedagainst each other. In one embodiment, said one or more inlet ports onsaid mating surface of said microfluidic device comprise dropletsprotruding above said mating surface and one or more outlet ports onsaid perfusion manifold comprise protruding droplets, such that slidingof step C) causes a droplet-to-droplet connection. In one embodiment,said carrier is engaged in a detachable fashion. In another embodiment,said carrier is engaged in a manner that is not detachable (e.g. onetime connection). In one embodiment, the assembly further comprising acover or lid for said fluid reservoirs, wherein said cover comprises aplurality of ports optionally associated with filters. In someembodiments, the cover ports comprise through-holes and filterspositioned above corresponding holes in a gasket. In some embodiments,the cover comprises one or more channels that route one or more of theports (such that the port is not a simple through-hole).

In one embodiment, the present invention contemplates a method ofperfusing cells, comprising: A) providing a) a perfusion manifoldassembly comprising i) one or more fluid reservoirs, ii) a fluidicbackplane positioned under, and in fluidic communication with, saidfluid reservoirs, said fluidic backplane comprising a fluid resistor andfluid channels that terminate at iii) a skirt having one or more fluidoutlet ports and one or more side tracks; and b) a microfluidic devicepositioned in a carrier, said carrier having one or more outer edges,said outer edges configured for detachably engaging said one or moreside tracks of said skirt, said microfluidic device comprising i) livingcells, and ii) microchannels in fluidic communication with ii) one ormore inlet ports on a iii) mating surface; B) positioning said carriersuch that said one or more outer edges engage said one or more sidetracks of said skirt; and C) sliding said carrier along said side trackuntil said one or more fluid inlet ports of said microfluidic device arepositioned against said one or more fluid outlet ports of said skirt ofsaid perfusion manifold assembly under conditions such that saidmicrofluidic device is linked and fluid flows from said fluid reservoirsof said perfusion manifold assembly through said one or more fluidoutlet ports into said one or more fluid inlet ports and into saidmicrochannels of said microfluidic device, thereby perfusing said cells.In one embodiment, said one or more inlet ports on said mating surfaceof said microfluidic device comprise droplets protruding above saidmating surface and one or more outlet ports on said skirt compriseprotruding droplets, such that sliding of step C) causes adroplet-to-droplet connection when one or more fluid inlet ports of saidmicrofluidic device are positioned against said one or more fluid outletports of said skirt of said perfusion manifold assembly.

In one embodiment, said droplet-to-droplet connection does not permitair to enter said one or more fluid inlet ports. In one embodiment, themating surface proximate to said droplets is hydrophobic.

In one embodiment, the method, further comprises the step of activatinga locking mechanism for restricting movement of the carrier. In oneembodiment, the method further comprises the step of placing saidperfusion manifold assembly with said linked microfluidic device in anincubator.

In one embodiment, the method (as described for any of the embodimentsof the perfusing method above) further comprises the step of placingsaid perfusion manifold assembly with said linked microfluidic deviceon, within or in contact with, a culture module. In one embodiment, saidfluid reservoirs of said perfusion manifold assembly are covered with acover assembly comprising a cover having a plurality ports, and saidculture module comprises a mating surface with pressure points thatcorrespond to the ports on the cover, such that the step of placing ofsaid perfusion manifold assembly with said linked microfluidic device inor on said culture module results in contact of said ports with saidpressure points. In one embodiment, said fluid reservoirs of saidperfusion manifold assembly are covered with a cover assembly comprisinga cover having a plurality ports, and said culture module comprises amating surface with pressure points that correspond to the ports on thecover, such that after the step of placing of said perfusion manifoldassembly with said linked microfluidic device in or on said culturemodule, the pressure points of the mating surface of the culture moduleare brought into contact with said through-holes of the cover assembly.In one embodiment, said fluid reservoirs of said perfusion manifoldassembly are covered with a cover assembly comprising a cover having aplurality of through-hole ports associated with filters andcorresponding holes in a gasket, and said culture module comprises amating surface with pressure points that correspond to the through-holeports on the cover, such that the step of placing of said perfusionmanifold assembly with said linked microfluidic device on said culturemodule results in contact of said through-holes with said pressurepoints. In one embodiment, the fluid reservoirs of said perfusionmanifold assembly are covered with a cover assembly comprising a coverhaving a plurality of through-hole ports associated with filters andcorresponding holes in a gasket, and said culture module comprises amating surface with pressure points that correspond to the through-holeports on the cover, such that after the step of placing of saidperfusion manifold assembly with said linked microfluidic device in oron said culture module, the pressure points of the mating surface of theculture module are brought into contact with said through-holes of thecover assembly.

In one embodiment, said culture module comprises volumetric controllers.In one embodiment, said volumetric controllers apply pressure to saidfluid reservoirs via said pressure points corresponding to said ports onsaid cover. In one embodiment, said culture module comprises pressureactuators. In one embodiment, said culture module comprises pressurecontrollers. In one embodiment, said pressure controllers apply pressureto said fluid reservoirs via said pressure points (e.g. on a pressuremanifold) corresponding to said ports (e.g. through-hole ports) on saidcover. In one embodiment, said culture module comprises a plurality ofperfusion manifold assemblies. In one embodiment, said culture modulecomprises integrated valves. In one embodiment, said integrated valvesare in a pressure manifold. In one embodiment, said valves compriseSchrader valves.

The present invention also contemplates the culture module as a device.In one embodiment, the device comprises an actuation assembly configuredto move a plurality of microfluidic devices (such as the perfusionmanifold assemblies described herein) against a pressure manifold, saidpressure manifold comprising integrated valves. In one embodiment, it isconfigured to move the microfluidic devices up against a non-movingpressure manifold. In one embodiment, the device comprises an actuationassembly configured to move one or more perfusion manifold assembliesinto contact with a pressure manifold. In one embodiment, the devicecomprises an actuation assembly configured to move a pressure manifold(up or down) into contact with the plurality of perfusion manifoldassemblies. In some embodiments, said pressure manifold comprisesintegrated valves and elastomeric membranes. In some embodiments, theelastic/pliable seal is disposed on the pod or lid and not on thepressure manifold. In either embodiment, the present invention is notintended to be limited to a membrane, since a membrane is only onespecific way to do this; in other embodiments, o-rings, gaskets (thickerthan a membrane), pliable materials, or vacuum grease are used instead.In one embodiment, the said valves comprise Schrader valves. In someembodiments, the pressure manifold is adapted to sense the presence of acoupled perfusion manifold assembly or microfluidic device, for example,in order to reduce the leakage of pressure or fluid in the absence of acoupled device. Importantly, the pressure manifold, in a preferredembodiment, takes the few pressure sources and disperses them to everyperfusion manifold assembly. In some embodiments, the pressure manifoldis also designed to directly align with the perfusion manifoldassemblies (e.g. via alignment features in the pressure manifold matingsurface). In one embodiment, the perfusion manifold assemblies slideinto alignment features on the bottom of the pressure manifold that makesure the seals in the pressure manifold are always aligned with theports on the perfusion manifold assemblies. In some embodiments, thepressure manifold has a set of springs that push down on the perfusionmanifold assemblies when the pressure manifold is actuated. Thesesprings force the lid up against the reservoir of the perfusion manifoldassembly to create the seal that holds pressure (and avoids leaks)within the perfusion manifold assembly when pressure is passed throughthe lid ports.

The present invention also contemplates the culture module and theperfusion disposables (PDs) as a system. In one embodiment, the systemcomprises a device comprising an actuation assembly configured to move aplurality of microfluidic devices (such as the perfusion manifoldassemblies described herein) against a pressure manifold, said pressuremanifold comprising integrated valves. In one embodiment, it isconfigured to move the microfluidic devices up against a non-movingpressure manifold. In one embodiment, the system comprises a) device,comprising an actuation assembly configured to move b) a plurality ofmicrofluidic devices (such as the perfusion disposables) into contactwith a pressure manifold. In one embodiment, the system comprises a)device, comprising an actuation assembly configured to move a pressuremanifold, said pressure manifold comprising integrated valves and seals(e.g. elastomeric membranes), said seals (e.g. elastomeric membranes) incontact with b) a plurality of microfluidic devices. In one embodiment,said microfluidic devices are perfusion disposables. In someembodiments, the elastic/pliable seal is disposed on the pod or lid andnot on the pressure manifold. In either embodiment, the presentinvention is not intended to be limited to a membrane, since a membraneis only one specific way to do this; in other embodiments, o-rings,gaskets (thicker than a membrane), pliable materials, or vacuum greaseare used instead. In one embodiment, said valves comprise Schradervalves. In one embodiment, the manifold uses a bi-stable engagementmechanism so that the actuator does not need to be always on to provideengagement and continuous pressure to the lid. In a bi-stable mechanism,the actuator engages the manifold and then can be turned off. This isuseful in situations where the actuator might generate excessive heatwhile powered for long periods of time. In one embodiment, the perfusiondisposable is engaged with a microfluidic chip. In one embodiment, themicrofluidic chip comprises a top channel, a bottom channel, and amembrane separating at least a portion of said top and bottom channels.In one embodiment, the microfluidic device comprises cells on themembrane and/or in or on the channels.

The present invention also contemplates drop-to-drop connection schemesfor putting a microfluidic device in fluidic communication with a fluidsource or another device, including but not limited to, putting amicrofluidic device in fluidic communication with the perfusion manifoldassembly. In one embodiment, the present invention contemplates afluidic device comprising a substrate having a first surface, said firstsurface comprising one or more fluidic ports, wherein said first surfaceis adapted to stably retain one or more liquid droplets comprising afirst liquid at the one or more fluidic ports. In one embodiment, saidfirst surface comprises one or more regions surrounding the one or morefluidic ports, and wherein said regions are adapted to resist wetting bysaid first liquid. In one embodiment, said regions are adapted to behydrophobic. In one embodiment, said one or more regions comprise afirst material selected to resist wetting by said first liquid. It isnot intended that the present invention be limited by any particularfirst material. However, in one embodiment, the first material isselected from the group consisting of poly-tetrafluoroethylene (PTFE), aperfluoroalkoxy alkane (PFA), fluorinated ethylenepropylene (FEP),polydimethylsiloxane (PDMS), nylon (some grades are hydrophilic and someare hydrophobic), polypropylene, polystyrene and polyimide. In oneembodiment, the substrate comprises said first material. In oneembodiment, said first material is bonded, adhered, coated or sputteredonto said first surface. In one embodiment, said first materialcomprises a hydrophobic gasket. In one embodiment, the one or moreregions are adapted to resist wetting by said first liquid by means ofplasma treatment, ion treatment, gas-phase deposition, liquid-phasedeposition, adsorption, absorption or chemical reaction with one or moreagents.

In one embodiment, said first surface comprises one or more regionssurrounding the one or more fluidic ports, and wherein said regions areadapted to promote wetting by said first liquid. In one embodiment, saidregions are adapted to be hydrophilic. In one embodiment, said one ormore regions comprise a first material selected to promote wetting bysaid first liquid. Again, it is not intended that the present inventionbe limited to any particular first material. However, in one embodiment,the first material is selected from the group consisting ofpolymethylmethacrylate (PMMA), polyvinyl alcohol (PVOH), polycarbonate(PC), polyether ether ketone (PEEK), polyethylene terephthalate (PET),polyfulfone, polystyrene, polyvinyl acetate (PVA), nylon, polyvinylfluoride (PVF), polyvinylidiene chloride (PVDC), polyvinyl chloride(PVC) and acrylonitrile-butadiene-styrene (ABS). In one embodiment, thesubstrate comprises said first material. In one embodiment, said firstmaterial is bonded, adhered, coated or sputtered onto said firstsurface. In one embodiment, said first material comprises a hydrophilicgasket. In one embodiment, the one or more regions are adapted topromote wetting by said first liquid by means of plasma treatment, iontreatment, gas-phase deposition, liquid-phase deposition, adsorption,absorption or chemical reaction with one or more agents.

In one embodiment, the first surface comprises one or more ridgessurrounding the one or more fluidic ports. In one embodiment, the firstsurface comprises one or more recesses surrounding the one or morefluidic ports. In one embodiment, said first surface is adapted tostably retain one or more aqueous liquid droplets. In one embodiment,said first surface is adapted to stably retain one or more non-aqueousliquid droplets. In one embodiment, said first surface is adapted tostably retain one or more oil droplets.

The present invention also contemplates systems comprising devices thatretain droplets. In one embodiment, the system comprises: a) a firstsubstrate comprising a first surface, said first surface comprising afirst set of one or more fluidic ports, wherein said first surface isadapted to stably retain one or more liquid droplets comprising a firstliquid at the first set of fluidic ports, b) a second substratecomprising a second surface, said second surface comprising a second setof one or more fluidic ports, and c) a mechanism for fluidicallycontacting (and connecting) the first set of fluidic ports to the secondset of fluidic ports.

The present invention also contemplates methods of retaining droplets sothat they can be combined to establish a fluidic connection. In oneembodiment, a method for establishing a fluidic connection iscontemplated, comprising: a) providing a first substrate comprising afirst surface, said first surface comprising a first set of one or morefluidic ports, wherein said first surface is adapted to stably retainone or more liquid droplets comprising a first liquid at the first setof fluidic ports, b) providing a second substrate comprising a secondsurface, said second surface comprising a second set of one or morefluidic ports, and c) contacting the first set of fluidic ports and thesecond set of fluidic ports (e.g. via a controlled engagement). In apreferred embodiment, the contacting of step c) comprises aligning thefirst set of fluidic ports and the second set of fluidic ports andbringing the aligned sets of ports into contact.

In one embodiment, the present invention contemplates systems andmethods where a microfluidic device is brought into contact with a fluidsource in a drop-to-drop connection. In one embodiment, the presentinvention contemplates a method, comprising: a) providing i) a fluidsource in fluidic communication with a first fluid port positioned on afirst mating surface, said first fluid port comprising a firstprotruding fluid droplet; ii) a microfluidic device comprising amicrochannel in fluidic communication with an second fluid port on asecond mating surface, said second fluid port comprising a secondprotruding fluid droplet; and b) bringing said first protruding fluiddroplet and said second fluid droplet together in a droplet-to-dropletconnection, so that fluid can flow from said fluid source through saidfirst fluid port into said second fluid port of said microfluidicdevice. In one embodiment, the present invention contemplates a system,comprising: a) a fluid source in fluidic communication with a firstfluid port positioned on a first mating surface, said first fluid portadapted to support a first protruding fluid droplet; b) a microfluidicdevice comprising a microchannel in fluidic communication with an secondfluid port on a second mating surface, said second fluid port adapted tosupport a second protruding fluid droplet; and c) a mechanism forbringing said first protruding fluid droplet and said second fluiddroplet together in a droplet-to-droplet connection, so that fluid canflow from said fluid source through said first fluid port into saidsecond fluid port of said microfluidic device. In one embodiment, thefirst protruding fluid droplet protrudes downward from said first matingsurface and said second protruding fluid droplet protrudes upward fromsaid second mating surface. In one embodiment, the first protrudingfluid droplet protrudes upward from said first mating surface and saidsecond protruding fluid droplet protrudes downward from said secondmating surface. In one embodiment, said mechanism lifts the secondmating surface upward into contact with said first mating surface. Inanother embodiment, said mechanism lifts the first mating surface upwardinto contact with said second mating surface. In still anotherembodiment, said mechanism lowers the second mating surface into contactwith said first mating surface. In yet another embodiment, saidmechanism lowers the first mating surface into contact with said secondmating surface.

In one embodiment, the present invention contemplates that droplets arecontrolled by surface treatments. In one embodiment of the system, saidfirst mating surface comprises a region surrounding said first fluidport, and wherein said region is adapted to resist wetting by saidfluid. In one embodiment said region is adapted to be hydrophobic. Inone embodiment, said region comprises a first material selected toresist wetting by said fluid. It is not intended that the presentinvention be limited by the nature of the first material. However, inone embodiment, the first material is selected from the group consistingof poly-tetrafluoroethylene (PTFE), a perfluoroalkoxy alkane (PFA),fluorinated ethylenepropylene (FEP), polydimethylsiloxane (PDMS), nylon(some grades are hydrophobic), polypropylene, polystyrene and polyimide.It is not intended that the present invention be limited by the natureby which the first material is attached to the surface. However, in oneembodiment, said first material is bonded, adhered, coated or sputteredonto said first mating surface. The present invention also contemplatesadding features with intrinsic hydrophobic surfaces, or surfaces thatcan be made hydrophobic. In one embodiment, said first materialcomprises a hydrophobic gasket. It is not intended that the presentinvention be limited by the particular treatment regime use to modifysurfaces, or regions of surfaces. However, in one embodiment, saidregion of said first mating surface is adapted to resist wetting bymeans of plasma treatment, ion treatment, gas-phase deposition,liquid-phase deposition, adsorption, absorption or chemical reactionwith one or more agents.

While an embodiment has been discussed above for adapting surfaces orregions of surfaces to resist wetting, the present inventioncontemplates embodiments wherein said first mating surface comprises aregion surrounding said first fluid port, and wherein said region isadapted to promote wetting by said fluid. In one embodiment, said regionis adapted to be hydrophilic. In one embodiment, said region comprises afirst material selected to promote wetting by said first liquid. It isnot intended that the present invention be limited to particular firstmaterials for promoting wetting. However, in one embodiment, the firstmaterial is selected from the group consisting of polymethylmethacrylate(PMMA), polyvinyl alcohol (PVOH), polycarbonate (PC), polyether etherketone (PEEK), polyethylene terephthalate (PET), polyfulfone,polystyrene, polyvinyl acetate (PVA), nylon (certain grades arehydrophilic), polyvinyl fluoride (PVF), polyvinylidiene chloride (PVDC),polyvinyl chloride (PVC) and acrylonitrile-butadiene-styrene (ABS). Itis also not intended that the present invention be limited by thetechnique for attaching the first material to the surface. However, inone embodiment, said first material is bonded, adhered, coated orsputtered onto said first mating surface. The present invention alsocontemplates introducing structures or features with intrinsichydrophilic surfaces, or surfaces that can be made hydrophilic. Forexample, in one embodiment, said first material comprises a hydrophilicgasket. It is also not intended that the present invention be limited tothe treatment regime for promoting wetting. For example, in oneembodiment, said region of said first mating surface is adapted topromote wetting by means of plasma treatment, ion treatment, gas-phasedeposition, liquid-phase deposition, adsorption, absorption or chemicalreaction with one or more agents.

The present invention also contemplates structures and geometricalfeatures that can be molded or formed as part of the surface, attachedto, deposited on, printed on or bonded to the sources, or machined into,etched into or ablated into the surface. For example, in one embodiment,the first mating surface comprises one or more ridges surrounding saidfirst fluid ports. In another embodiment, the first mating surfacecomprises one or more recesses surrounding said first fluid port.

The present invention is also not limited to drop-to-drop connectionswith only aqueous fluids. While in one embodiment, said first matingsurface is adapted to stably retain an aqueous protruding fluid droplet,in another embodiment, said first mating surface is adapted to stablyretain a non-aqueous protruding fluid droplet, including but not limitedto an oil protruding droplet.

The present invention also contemplates method for merging dropletsusing a drop-to-drop scheme. In one embodiment, the present inventioncontemplates a method of merging droplets, comprising: a) providing i) afluid source in fluidic communication with a first fluid port positionedon a first mating surface, said first fluid port comprising a firstprotruding fluid droplet; and ii) a microfluidic device or chipcomprising a microchannel in fluidic communication with a second fluidport on a second mating surface, said second fluid port comprising asecond protruding fluid droplet; and b) bringing said first protrudingfluid droplet and said second fluid droplet together in adroplet-to-droplet connection, whereby the first and second fluiddroplets merge so that fluid flows from said fluid source through saidfirst fluid port into said second fluid port of said microfluidicdevice. In one embodiment, the microfluidic chip comprises a topchannel, a bottom channel, and a membrane separating at least a portionof said top and bottom channels. In one embodiment, the microfluidicdevice comprises cells on the membrane and/or in or on the channels. Itis not intended that the present invention be limited to particularorientations or the two mating surfaces. In one embodiment, the firstprotruding fluid droplet protrudes downward from said first matingsurface and said second protruding fluid droplet protrudes upward fromsaid second mating surface. In another embodiment, the first protrudingfluid droplet protrudes upward from said first mating surface and saidsecond protruding fluid droplet protrudes downward from said secondmating surface. It is also not intended that the present invention belimited by how the droplets are brought together. In one embodiment,step b) comprises lifting the second mating surface upward into contactwith said first mating surface. In another embodiment, step b) compriseslifting the first mating surface upward into contact with said secondmating surface. In yet another embodiment, step b) comprising loweringthe second mating surface into contact with said first mating surface.In still another embodiment, step b) comprises lowering the first matingsurface into contact with said second mating surface. In a preferredembodiment, said droplet-to-droplet connection does not permit air toenter said fluid inlet port.

The present invention contemplates surface treatments to promotewetting. In one embodiment, said first mating surface comprises a regionsurrounding said first fluid port, wherein said region is adapted topromote wetting by said fluid. In one embodiment, said region is adaptedto be hydrophilic. In one embodiment, said region comprises a firstmaterial selected to promote wetting by said fluid. While not intendedto limit the invention to any particular first material, in oneembodiment, the first material is selected from the group consisting ofpolymethylmethacrylate (PMMA), polyvinyl alcohol (PVOH), polycarbonate(PC), polyether ether ketone (PEEK), polyethylene terephthalate (PET),polyfulfone, polystyrene, polyvinyl acetate (PVA), nylon, polyvinylfluoride (PVF), polyvinylidiene chloride (PVDC), polyvinyl chloride(PVC) and acrylonitrile-butadiene-styrene (ABS). While not intending tolimit the invention to any particular attachment approach, in oneembodiment, said first material is bonded, adhered, coated or sputteredonto said first mating surface.

In some embodiments, the present invention contemplates adding featuresor structures to a surface, including structures with intrinsicallyhydrophilic surfaces (or surfaces that can be made hydrophilic). In oneembodiment, said first material comprises a hydrophilic gasket.

It is not intended that the present invention be limited to anyparticular surface treatment technique. However, in one embodiment, saidregion of said first mating surface is adapted to promote wetting bymeans of plasma treatment, ion treatment, gas-phase deposition,liquid-phase deposition, adsorption, absorption or chemical reactionwith one or more agents.

Additional structures can be molded or otherwise formed into or on tothe surfaces. For example, in one embodiment, the first mating surfacecomprises one or more ridges surrounding said first fluid port. Inanother embodiment, the first mating surface comprises one or morerecesses surrounding said first fluid port.

As noted above, the fluid need not be an aqueous fluid. While in oneembodiment, the present invention contemplates said first mating surfaceis adapted to stably retain an aqueous protruding fluid droplet, inanother embodiment, said first mating surface is adapted to stablyretain a non-aqueous protruding fluid droplet, including but not limitedto retaining an oil protruding droplet.

The present invention also contemplates systems for linking portstogether. In one embodiment, the system comprises: a) a first substratecomprising a first fluidic port, b) a second substrate comprising asecond fluidic port, c) a guide mechanism adapted to align the firstport and the second port, and (optionally) d) a retention mechanismadapted to retain the first substrate in contact with the secondsubstrate. While not intending to limiting the invention to anyparticular guide mechanism, in one embodiment, the guide mechanism is aguide track positioned on said first substrate, said guide trackconfigured to engage a portion of said second substrate. While thepresent invention contemplates embodiments wherein the retentionmechanism is on the first or second substrate, in one embodiment, theretention mechanism is a clip positioned on said second substrate, saidclip configured to engage said first substrate.

In another embodiment, the present invention contemplates a systemcomprising: a) a first substrate comprising a first set of one or morefluidic ports, b) a second substrate comprising a second set of one ormore fluidic ports, c) a guide mechanism adapted to align the first setof ports and the second set of ports, and d) a retention mechanismadapted to retain the first substrate in contact with the secondsubstrate. Again, a variety of guide mechanisms are contemplated (anddiscussed herein). In one embodiment, the guide mechanism comprises aguide shaft or a hole, groove, orifice or other cavity configured toaccept a guide shaft. However, in one embodiment, the guide mechanism isa guide track positioned on said first substrate, said guide trackconfigured to engage a portion of said second substrate. Again, avariety of retention mechanisms are contemplated (and described herein).However, in one embodiment, the retention mechanism is a clip positionedon said second substrate, said clip configured to engage said firstsubstrate.

The present invention also contemplates methods for linking ports in amanner such that a fluidic connection is established. In one embodiment,the present invention contemplates a method for establishing a fluidicconnection, comprising: a) providing a first substrate comprising afirst fluidic port, a second substrate comprising a second fluidic port,and a guide mechanism adapted to guide the second substrate, b) engagingthe second substrate with the guide mechanism, c) aligning the first andsecond sets of fluidic ports by help of the guide mechanism, and d)contacting the first and second fluidic ports to establish a fluidicconnection. While a variety of guide mechanisms are contemplated, in oneembodiment, said guide mechanism comprises a guide track positioned onsaid first substrate, said guide track configured to engage a portion ofsaid second substrate. In one embodiment of this method for establishinga fluidic connection, said second substrate comprises a microfluidicdevice comprising a mating surface, wherein said second fluidic port ispositioned on said mating surface and comprises a droplet protrudingabove said mating surface. In a further embodiment, said first substratecomprises a mating surface, wherein said first fluidic port ispositioned on said mating surface and comprises a protruding droplet.Still further in this embodiment, said contacting of step d) causes adroplet-to-droplet connection when said first and second fluidic portsto establish a fluidic connection. It is preferred that saiddroplet-to-droplet connection does not permit air to enter said one ormore fluid inlet ports. While the present invention is not limited tothe manner of aligning, in one embodiment, said aligning of step c)comprises sliding the second substrate by means of the guide track.While a variety of designs and conformations for the guide track arecontemplated, in one embodiment, said guide track comprises first andsecond sections, said first section shaped to support the aligning ofstep c), said second section shaped to support the contacting of stepd).

While the present invention contemplates embodiments where the retentionmechanism is on the first substrate, in one embodiment, said secondsubstrate comprises a retention mechanism adapted to retain the firstsubstrate in contact with the second substrate. In some embodiments, theretention mechanism automatically engages when the first and secondsubstrates make contact and establish a fluidic connection. However, inone embodiment, the present invention contemplates the active step of e)activating the retention mechanism.

While two substrate systems have been described above, the presentinvention also contemplates three substrate systems. In one embodiment,the system comprises: a) a first substrate comprising a first fluidicport, b) a second substrate comprising a second fluidic port, c) a thirdsubstrate configured to support said second substrate; d) a guidemechanism adapted to align the first port with second port, and e) aretention mechanism means adapted to retain the first substrate incontact with the second substrate.

As noted previously, a variety of guide mechanisms are contemplated (anddescribed herein). In one embodiment, the guide mechanism comprises aguide shaft or a hole, groove, orifice or other cavity configured toaccept a guide shaft. One or more guide shafts or other projections canbe on one substrate, with one or more holes, grooves, orifices or othercavities on the other substrate configured to accept the one or moreguide shafts or other projections. In one embodiment, the guidemechanism comprises a guide track. The guide track(s) can be in anyorientation (e.g. coming from above rather than from either side). Whilethe present invention contemplates that the guide mechanism might beattached to the first, second or third substrate, in one embodiment, theguide track is positioned on said first substrate. While the presentinvention contemplates embodiments wherein either the second or thirdsubstrates are have features or structures configured to engage theguide mechanism, in one embodiment, the present invention contemplatesthat the third substrate comprises edges configured to engage said guidetrack. In one embodiment, the second substrate comprises edgesconfigured to engage said guide track. While the present inventioncontemplates embodiments wherein the retention mechanism is positionedon the first or second substrates, in one embodiment, said retentionmechanism is positioned on said third substrate. As noted previously, avariety of retention mechanisms are contemplated. In one embodiment,said retention mechanism comprises a clip configured to engage saidfirst substrate. In another embodiment, said retention mechanismcomprises a clamp configured to engage said first substrate underconditions such that contact between said first and second substrates ismaintained. In yet another embodiment, said retention mechanismcomprises a stud configured to engage a hole on said first substrate. Instill another embodiment, said retention mechanism engages a portion ofsaid first substrate in a friction fit. In one embodiment, saidretention mechanism is selected from the group consisting of an adhesive(including a laminate), a heat stake, and a screw.

While the present invention contemplates systems wherein the componentsof the systems are described (see above), the present invention alsocontemplates assemblies, where the components are arranged, attached orconnected in certain ways. In one embodiment, the present inventioncontemplates an assembly, comprising: a) a first substrate comprising afirst fluidic port and a guide mechanism, said first substratepositioned against and in contact with b) a second substrate comprisinga second fluidic port, wherein said first and second ports are alignedso as to permit fluidic communication, said second substrate supportedby c) a carrier, said carrier comprising a portion engaging said guidemechanism of said first substrate. While the present inventioncontemplates embodiments, where a retention mechanism is positioned onsaid first or second substrate, in one embodiment, said carrier furthercomprises a retention mechanism for retaining said contact between saidfirst and second substrates. While a variety of guide mechanisms arecontemplated (and described herein), in one embodiment, the guidemechanism comprises a guide track. The present invention is not limitedto a single guide track; two or more guide tracks may be employed. Forexample, in one embodiment the guide track is positioned on one or moresides of said first substrate. In one embodiment, the carrier portionengaging said first substrate comprises one or more edges configured toengage said guide track.

While a variety of retention mechanisms are contemplated (and describedherein) in one embodiment of the assembly, said retention mechanismcomprises a clip configured to engage said first substrate. In anotherembodiment, said retention mechanism comprises a clamp configured toengage said first substrate. In yet another embodiment, said retentionmechanism comprises a stud configured to engage a hole on said firstsubstrate. In a particular embodiment, said retention mechanism engagesa portion of said first substrate in a friction fit. In one embodiment,said retention mechanism is selected from the group consisting of anadhesive (including but not limited to a laminate), a heat stake, and ascrew.

The present invention also contemplates methods for establishing afluidic connection by bringing fluidic ports together where threesubstrates are involved. In one embodiment, the present inventioncontemplates a method for establishing a fluidic connection, comprising:a) providing: a first substrate comprising a first fluidic port, asecond substrate comprising a second fluidic port, a third substrateconfigured to support said second substrate, and a guide mechanism; b)aligning said first and second ports with said guide mechanism; and c)contacting said first port with said second port under conditions suchthat a fluidic connection is established between said first and secondsubstrate. In one embodiment of this three substrate method, said secondsubstrate comprises a microfluidic device comprising a mating surface,wherein said second fluidic port is positioned on said mating surfaceand comprises a droplet protruding above said mating surface. Further inthis embodiment, said first substrate comprises a mating surface,wherein said first fluidic port is positioned on said mating surface andcomprises a protruding droplet. Still further in this embodiment, saidcontacting of step c) causes a droplet-to-droplet connection when saidfirst and second fluidic ports to establish a fluidic connection. It ispreferred that said droplet-to-droplet connection does not permit air toenter said one or more fluid inlet ports.

Again, a variety of guide mechanisms are contemplated and describedherein. In one embodiment, the guide mechanism comprises a guide track.While the present invention contemplates positioning the guide track onsaid first, second or third substrates, in a preferred embodiment, theguide track is positioned on said first substrate. In one embodiment,the third substrate comprises edges configured to engage said guidetrack. While not intending that the invention be limited to theparticular technique for aligning, in one embodiment, the presentinvention contemplates said aligning of step b) comprises sliding saidthird substrate by means of said guide track. In one embodiment, saidguide track comprises first and second sections, said first sectionshaped to support the aligning of step b), said second section shaped tosupport the contacting of step c). In one embodiment, said first sectionis linear and said second section is curved. In yet another embodiment,said guide mechanism comprises a mechanism on which said third substraterotates or pivots during step d). For example, in one embodiment, saidguide mechanism comprises a hinge, joint, or pivot point.

While the present invention contemplates embodiments where the retentionmechanism is positioned on the first or second substrates, in oneembodiment, the present invention contemplates that said third substratefurther comprises a retention mechanism for retaining alignment of saidfirst and second ports. Again, a variety of retention mechanisms arecontemplated. In one embodiment, said retention mechanism comprises aclip configured to engage said first substrate. In one embodiment, saidretention mechanism comprises a clamp configured to engage said firstsubstrate under conditions such that contact between said first andsecond substrates is maintained. In yet another embodiment, saidretention mechanism comprises a stud configured to engage a hole on saidfirst substrate. In still another embodiment, said retention mechanismengages a portion of said first substrate in a friction fit. In oneembodiment, said retention mechanism is selected from the groupconsisting of an adhesive (including but not limited to a laminate), aheat stake, and a screw. The present invention also contemplatesembodiments wherein the third substrate is a carrier for the secondsubstrate.

In one embodiment, the present invention contemplates a method forestablishing a fluidic connection, comprising: a) providing: a firstsubstrate comprising a guide mechanism and a first fluidic port on afirst mating surface, a second substrate comprising a second fluidicport on a second mating surface and a bottom surface, and a carrier incontact with said bottom surface of said second substrate, said carriercomprising a retention mechanism and one or more edges for engaging saidguide mechanism; b) engaging said guide mechanism of said firstsubstrate with one or more edges of said carrier; c) aligning said firstand second ports with said guide mechanism; d) contacting said firstmating surface with said second mating surface under conditions suchthat said first port contacts said second port and a fluidic connectionis established between said first and second substrate. In oneembodiment of this method, said second fluidic port comprises a dropletprotruding above said mating surface of said second substrate. In oneembodiment, said first fluidic port comprises a protruding droplet. Inone embodiment, said contacting of step d) causes a droplet-to-dropletconnection when said first and second fluidic ports to establish afluidic connection. It is preferred that said droplet-to-dropletconnection does not permit air to enter said one or more fluid inletports. While a variety of guide mechanisms are contemplated, in oneembodiment, the guide mechanism comprises a guide track. The presentinvention is not limited to embodiments where there is only one guidetrack; two or more guide tracks may be used. In one embodiment, theguide track is positioned on one or more sides of said first substrate.In a preferred embodiment, the carrier comprises one or more edgesconfigured to engage said guide track. While a variety of aligningapproaches are contemplated, in one embodiment, said aligning of step c)comprises sliding said carrier by means of said guide track. While avariety of designs and configurations for the guide track arecontemplated, in one embodiment, said guide track comprises first andsecond sections, said first section shaped to support the aligning ofstep c), said second section shaped to support the contacting of stepd). In one embodiment, said first section is linear and said secondsection is curved. In yet another embodiment, said guide mechanismcomprises a mechanism on which said carrier rotates or pivots duringstep d). In this embodiment, said guide mechanism may comprise a hinge,a joint, a socket or other pivot point.

In some embodiments, the retention mechanism automatically engages whenor after contact is made in step d). However, in one embodiment, thepresent invention contemplates the active step of e) activating saidretention mechanism under condition such that said alignment of saidfirst and second ports is retained. Again, a variety of retentionmechanisms are contemplated. In one embodiment, said retention mechanismcomprises a clip configured to engage said first substrate. In oneembodiment, said retention mechanism comprises a clamp configured toengage said first substrate under conditions such that contact betweensaid first and second substrates is maintained. In one embodiment, saidretention mechanism comprises a stud configured to engage a hole on saidfirst substrate. In one embodiment, said retention mechanism engages aportion of said first substrate in a friction fit. In one embodiment,said retention mechanism is selected from the group consisting of anadhesive (including but not limited to a laminate, a heat stake, and ascrew).

The present invention also contemplates devices for perfusing cells,including devices that apply pressure to fluid reservoirs to create aflow of fluid (e.g. culture media). The present invention contemplates,in one embodiment, a device, comprising an actuation assembly configuredto move a pressure manifold, said pressure manifold comprisingintegrated valves. In one embodiment, said device further compriseselastomeric membranes. In one embodiment, said valves comprise Schradervalves. In one embodiment, said pressure manifold comprises a matingsurface with pressure points. In one embodiment, the device furthercomprises pressure controllers. In one embodiment, said pressurecontrollers are configured to apply pressure via said pressure points.In one embodiment, said actuation assembly comprises pneumatic cylinderoperably linked to said pressure manifold. In one embodiment, saidmating surface further comprises alignment features configured to aligna microfluidic device or chip when said microfluidic device or chipengages said mating surface. In one embodiment, said device is a culturemodule for perfusing cells. In one embodiment, the microfluidic chip isengaged with a perfusion manifold assembly (and the alignment featuresare configured to align the perfusion manifold assembly). In oneembodiment, the microfluidic chip comprises a top channel, a bottomchannel, and a membrane separating at least a portion of said top andbottom channels. In one embodiment, the microfluidic device comprisescells on the membrane and/or in or on the channels.

The present invention also contemplates systems where a device fordelivering pressure is linked to a plurality of microfluidic devices,and more preferably, the plurality of microfluidic devices (such as thevarious embodiments of the perfusion disposables discussed herein) aresimultaneously linked (although they can be linked individually orsequentially, if desired). In one embodiment, the present inventioncontemplates a system, comprising a) device comprising an actuationassembly configured to move a pressure manifold, said pressure manifoldcomprising integrated valves, said pressure manifold in contact with b)a plurality of microfluidic devices (such as the various embodiments ofthe perfusion disposables discussed herein). In one embodiment, saidpressure manifold further comprises elastomeric membranes, and saidelastomeric membranes are in contact with said microfluidic devices. Inone embodiment, said microfluidic devices are perfusion disposables. Inone embodiment, said valves comprise Schrader valves. In one embodiment,each of said microfluidic devices is covered with a cover assemblycomprising a cover having a plurality of ports, and said pressuremanifold comprising a mating surface with pressure points thatcorrespond to the ports on the cover, wherein the pressure points of themating surface of the pressure manifold are in contact with said portsof the cover assembly. In one embodiment, said ports comprisethrough-hole ports associated with filters and corresponding holes in agasket. In one embodiment, the device further comprises pressurecontrollers. In one embodiment, said pressure controllers are configuredto apply pressure via said pressure points. In one embodiment, saidactuation assembly comprises pneumatic cylinder operably linked to saidpressure manifold. In one embodiment, said mating surface of thepressure manifold further comprises alignment features configured toalign a microfluidic device when said microfluidic device engages saidmating surface. In a preferred embodiment, said device is a culturemodule for perfusing cells. In one embodiment of such a culture module,the culture module is configured to accept one or more trays, each traycomprising a plurality of microfluidic devices. In one embodiment, theculture module further comprises a user interface to control saidculture module. In one embodiment, each tray comprising a plurality ofperfusion manifold assemblies. In one embodiment, a microfluidic chip isengaged with each perfusion manifold assembly (and the alignmentfeatures of the pressure manifold mating surface are configured to aligneach perfusion manifold assembly). In one embodiment, the microfluidicchip comprises a top channel, a bottom channel, and a membraneseparating at least a portion of said top and bottom channels. In oneembodiment, the microfluidic device comprises cells on the membraneand/or in or on the channels.

The present invention also contemplates methods for perfusing cells(e.g. cells in microchannels of a microfluidic device, such as thevarious embodiments of the perfusion disposable discussed herein, wherecells were first seeded into said microfluidic device, with or without aseeding guide of the type described herein) with a culture module. Inone embodiment, the present invention contemplates a method of perfusingcells, comprising: A) providing a) a culture module, said culture modulecomprising i) an actuation assembly configured to move a plurality ofmicrofluidic devices against ii) a pressure manifold, said pressuremanifold comprising a mating surface with pressure points; and b) aplurality of microfluidic devices, each of said microfluidic devicescomprising i) one or more microchannels comprising living cells, ii) oneor more reservoirs comprising culture media, and iii) a cover assemblyabove said one or more reservoirs, said cover assembly comprising acover with ports that correspond to the pressure points on the pressuremanifold mating surface; B) placing said plurality of microfluidicdevices on or in said culture module; and C) simultaneously (orsequentially) contacting said ports on the cover of each microfluidicdevice of said plurality of microfluidic devices with said matingsurface of said pressure manifold, such that the ports are in contactwith said pressure points, under conditions such that culture mediaflows from said reservoirs into said microchannels of said microfluidicdevices, thereby perfusing said cells. In one embodiment, said pluralityof microfluidic devices are positioned on one or more trays prior tostep B) and said placing of step B) comprising moving at least a subsetof said plurality of microfluidic devices simultaneously into saidculture module. In one embodiment, said simultaneous contacting of stepC) is achieved by moving, via the actuation assembly, the plurality ofmicrofluidic devices up against the mating surface of the pressuremanifold. In another embodiment, the present invention contemplates amethod of perfusing cells, comprising: A) providing a) a culture module,said culture module comprising i) an actuation assembly configured tomove ii) a pressure manifold, said pressure manifold comprising a matingsurface with pressure points; and b) a plurality of microfluidicdevices, each of said microfluidic devices comprising i) one or moremicrochannels comprising living (viable) cells, ii) one or morereservoirs comprising culture media, and iii) a cover assembly abovesaid one or more reservoirs, said cover assembly comprising a cover withports that correspond to the pressure points on the pressure manifoldmating surface; B) placing said plurality of microfluidic devices on orin said culture module; and C) simultaneously (or sequentially)contacting said ports on the cover of each microfluidic device of saidplurality of microfluidic devices with said mating surface of saidpressure manifold, such that the ports are in contact with said pressurepoints, under conditions such that culture media flows from saidreservoirs into said microchannels of said microfluidic devices, therebyperfusing said cells. In the above embodiment, the plurality ofmicrofluidic devices are simultaneously linked. Thereafter, they can besimultaneously de-linked or disconnected from the pressure manifold. Inone embodiment, said plurality of microfluidic devices are positioned onone or more trays (or nests) prior to step B) and said placing of stepB) comprising moving at least a subset (at least three) of saidplurality of microfluidic devices simultaneously into said culturemodule. In one embodiment, said simultaneous contacting of step C) isachieved by moving, via the actuation assembly, the mating surface ofthe pressure manifold down onto said cover assemblies of said pluralityof microfluidic devices. In one embodiment of the perfusion method, themicrofluidic device comprises a microfluidic chip (including but notlimited to the microfluidic chip shown in FIG. 3A, with one or moremicrochannels and ports) engaged in a perfusion manifold assembly, theassembly comprising i) a cover or lid configured to serve as the top ofii) one or more fluid reservoirs, iii) a fluidic backplane under, and influidic communication with, said fluid reservoir(s), and iv) aprojecting member or skirt that engages the microfluidic chip (directly)or (indirectly through) a carrier containing the microfluidic chip. Itis preferred that the perfusing is done at a rate that results in (ormaintains) greater than 80%, and more preferably greater than 90%, andmost preferably, greater than 95% viability of the cells containedwithin the microfluidic chip. In one embodiment, the assembly furthercomprises a capping layer under said fluid reservoir(s). In oneembodiment, said fluidic backplane comprises a resistor. In a preferredembodiment, the microfluidic chip environment is maintained to besterile during said perfusing.

The present invention also contemplates controlling pressure whileperfusing cells (e.g. cells in microchannels of a microfluidic device,such as the various embodiments of the perfusion disposable discussedherein, where cells were first seeded into said microfluidic device,with or without a seeding guide of the type described herein), includingcontrolling pressure, in one embodiment, such that it is reliablymaintained at 1 pKa (plus or minus 0.5 pKa, and more preferably, plus orminus 0.15 pKa). In one embodiment, the present invention contemplates amethod of controlling pressure while perfusing cells, comprising: A)providing a) a plurality of microfluidic devices, each of saidmicrofluidic devices comprising i) one or more microchannels comprisingliving cells, ii) one or more reservoirs comprising culture media, b)one or more pressure actuators, B) coupling said pressure actuators toat least one of the said reservoirs, the coupling adapted such thatactuated pressure modulates the perfusion of at least some of saidliving cells, C) turning said one or more pressure actuators between twoor more pressure setpoints, thereby controlling pressure while perfusingsaid cells. In another embodiment, the present invention contemplates amethod of controlling pressure while perfusing cells, comprising: A)providing a) a culture module, said culture module comprising i) anactuation assembly configured to move ii) a pressure manifold, saidpressure manifold comprising a mating surface with pressure points, andiii) one or more pressure controllers to provide pressure to saidpressure points; and b) a plurality of microfluidic devices, each ofsaid microfluidic devices comprising i) one or more microchannelscomprising living cells, ii) one or more reservoirs comprising culturemedia, and iii) a cover assembly above said one or more reservoirs, saidcover assembly comprising a cover with ports that correspond to thepressure points on the pressure manifold mating surface; B) placing saidplurality of microfluidic devices on or in said culture module; C)simultaneously contacting said ports on the cover of each microfluidicdevice of said plurality of microfluidic devices with said matingsurface of said pressure manifold, such that the ports are in contactwith said pressure points, under conditions such that culture mediaflows from said reservoirs into said microchannels of said microfluidicdevices, thereby perfusing said cells; and D) turning (or switching)said one or more pressure controllers off for a period of time and onfor a period of time (or turning them between two or more setpoints),thereby controlling pressure while perfusing said cells. In oneembodiment, the switching is between setpoints 1 kPa and 0.5 kPa to getgood resolution within that range. In one embodiment, the switching isat three levels: 2 kPa, 1 kPa and 0 kPa for some advanced method. In oneembodiment, said pressure controllers are turned off and on (or betweensetpoints) in a switching pattern (e.g. they are turned off and on, orbetween setpoints, repeatedly at defined intervals). In a preferredembodiment, the switching pattern is selected such that the averagevalue of pressure acting liquid in said one or more reservoirscorresponds to a desired value. For cells, the desired value istypically low. For example, in one embodiment, the switching pattern isselected such that the average gas pressure is maintained below 1 kPa.In one embodiment of the method of perfusing and controlling pressure,the microfluidic device comprises a microfluidic chip (including but notlimited to the microfluidic chip shown in FIG. 3A, with one or moremicrochannels and ports) engaged in a perfusion manifold assembly, theassembly comprising i) a cover or lid configured to serve as the top ofii) one or more fluid reservoirs, iii) a fluidic backplane under, and influidic communication with, said fluid reservoir(s), and iv) aprojecting member or skirt that engages the microfluidic chip (directly)or (indirectly through) a carrier containing the microfluidic chip. Itis preferred that the perfusing is done at a rate that results ingreater than 80%, and more preferably greater than 90%, and mostpreferably, greater than 95% viability of the cells contained within themicrofluidic chip. In one embodiment, the assembly further comprises acapping layer under said fluid reservoir(s). In one embodiment, saidfluidic backplane comprises a resistor. In one embodiment, the ports onthe cover or lid are associated with filters. In one embodiment, thefilters are 0.2 micron, 0.4 micron or 25 micron filters. In a preferredembodiment, the microfluidic chip environment is maintained to besterile during said perfusing. In one embodiment, cycling the pressureregulators on and off brings the average value of pressure close to thedesired value, but the max and min values seen by the microfluidicdevice or chip are brought much closer to the desired value byincorporating the resistive filter at the inlet in the lid of theperfusion manifold assembly.

A pressure lid is contemplated as a device that allows for thepressurization of one or more fluid sources (e.g. reservoirs) within orotherwise associated with a microfluidic device. The present inventioncontemplates, in one embodiment, a pressure lid comprising a pluralityof ports configured to engage a pressure manifold. In one embodiment,the ports are associated with filters. In one embodiment, the lid isassociated with a gasket. In one embodiment, the pressure lid is movableor removably attached to a microfluidic device to allow improved accessto elements (e.g. reservoirs) within. In one embodiment, the presentinvention contemplates a method comprising a) providing a pressure lid,a microfluidic device comprising a fluid source, and a pressuremanifold, wherein the pressure lid comprising a plurality of portsconfigured to engage a pressure manifold; b) positioning said pressurelid over said fluid source so as to create a positioned pressure lid;and c) engaging said positioned pressure lid with said pressure manifoldunder conditions such that pressure is applied through said ports suchthat fluid from said fluid source moves into or through saidmicrofluidic device. In one embodiment, the method further comprising d)disengaging said positioned pressure lid from said pressure manifold.Thereafter, the pressure lid can be (optionally) removed and themicrofluidic device can be used without the lid.

The present invention also contemplates a system comprising: a)instrument for interfacing with b) a microfluidic device, saidmicrofluidic device either comprising or in fluidic communication withi) one or more fluid reservoirs and ii) a pressure lid comprising one ormore instrument-interface ports and one or more reservoir-interfaceports, wherein the pressure lid is adapted to convey pressure between atleast one of the instrument-facing ports and at least one of thereservoir-facing ports. In one embodiment, the instrument comprises a(moving or non-moving) pressure manifold. In one embodiment, the one ormore fluid reservoirs are disposed in a cartridge, said cartridge influidic communication with said microfluidic device. In one embodiment,the one or more fluidic reservoirs are disposed in the said microfluidicdevice.

The present invention also contemplates, as a device, a pressure lidcomprising one or more instrument-interface ports and one or morereservoir-interface ports, wherein the pressure lid is adapted to conveypressure between at least one of the instrument-facing ports and atleast one of the reservoir-facing ports, and wherein the pressure lid isadapted to form a pressure interface with at least one fluid reservoir.

The present invention also contemplates, as a device, a pressure lidcomprising one or more channels, each channel comprising aninstrument-interface end and anreservoir-interface end, the channelconfigured to convey pressure between an instrument and a fluidreservoir.

DESCRIPTION OF THE FIGURES

FIG. 1A is an exploded view of one embodiment of the perfusion manifoldassembly showing the cover (or cover assembly) off of the reservoirs(the reservoir body can be made of acrylic, for example), the reservoirspositioned above the backplane, the backplane in fluidic communicationwith the reservoirs, the skirt with a side track for engaging arepresentative microfluidic device or “chip” (which can be fabricatedout of plastic, such as PDMS, for example) having one or more inlet,outlet and (optional) vacuum ports, and one or more microchannels, thechip shown next to (but not in) one embodiment of a chip carrier (whichcan be fabricated out of a thermoplastic polymer, such as acrylonitrilebutadiene styrene (ABS), for example), the carrier being configured tosupport and carrier the chip, e.g. dimensioned so that the chip fitswithin a cavity. FIG. 1B shows the same embodiment of the perfusionmanifold assembly with the cover on and over the reservoirs, and thechip inside the chip carrier fully linked to the skirt of the perfusionmanifold assembly, and thereby in fluidic communication with thereservoirs. In one embodiment, each chip has two inputs, two outputs and(optionally) two connections for the vacuum stretch. In one embodiment,putting the chip in fluidic communication connects all six in oneaction, rather than connecting them one at a time. FIG. 1C is anexploded view of one embodiment of the perfusion manifold assembly(before the components have been assembled) comprising reservoirspositioned over a fluidic backplane (comprising a fluid resistor), thatis fluidically sealed with a capping layer and is positioned over askirt, with each piece dimensioned to fit over the next. In oneembodiment, the skirt comprises structure (e.g. made of polymer) thatborders or defines two open spaces, one of the spaces configured toreceive the carrier with the chip inside. In one embodiment, the skirthas structure that completely surrounds one open space and two “arms”that extend outwardly that define a second open space for receiving thecarrier. In one embodiment, the two arms have side tracks for slidablyengaging the carrier edges.

FIG. 2A is an exploded view of one embodiment of the cover assemblycomprising a pressure cover or pressure lid. In the illustratedembodiment, the pressure lid comprises a plurality of ports (e.g.through-hole ports) associated with filters and corresponding holes in agasket. The illustrated design of the holes in the gasket is intended topermit the gasket to aid in retaining the illustrated filters inposition. In alternative embodiments, gasket openings may employ a shapedifferent from openings in the lid. For example, the gasket can beshaped to follow the contour of one or more reservoirs with which it isintended to form a fluidic or pressure seal. In some embodiments, aplurality of gaskets may be employed. In some embodiments, the filtersand/or gasket may be fixed using an adhesive, heat stacking, bonding(ultrasonic, solvent-assisted, laser welding), clamped, or captured byelements of the lid and/or an additional substrate. Although theillustrated pressure lid comprises through-hole ports, alternativeembodiments comprise one or more channels that route at least onetop-surface port to one or more bottom surface ports, which need not bedirectly underneath the top-surface port. FIG. 2B shows the sameembodiment of the cover assembly illustrated in FIG. 2A with the filtersand gasket positioned within (and under) the cover. FIG. 2C-1 is across-section view of one embodiment of the cover assembly showing theridges or sealing tooth that surrounds both the through-hole ports inthe cover. FIG. 2C-2 is a magnified view of one portion of FIG. 2C-1(circled). In the illustrated embodiment, the cross section shape of thesealing tooth is a trapezoidal shape, but other contemplated embodimentsemploy other tooth shapes including but not limited to semi-circular,rectangular, polygonal, and triangular. FIG. 2D is a top view of oneembodiment of the reservoir chamber-cover assembly seal showing thesealing tooth, vacuum chamber and inlet and outlet chambers. FIG. 2E-1is a cross-section view of one embodiment of the cover assembly seal inconnection with the reservoir, showing the cover gasket and sealingtooth. FIG. 2E-2 is a magnified view of one portion of FIG. 2E-1(circled). As the pressure manifold (discussed below) engages the coverassembly, the pressure drives the cover assembly (including the covergasket) onto the sealing tooth, forming seals between each of thereservoir chambers.

FIG. 3A shows one embodiment of the microfluidic device or chip, showingtwo channels, each with an inlet and outlet port, as well as (optional)vacuum ports. FIG. 3B is a topside schematic of an alternativeembodiment of the perfusion disposable or “pod” featuring thetransparent (or translucent) cover over the reservoirs, with the chipinserted. The chip can be seeded with cells and then placed in a carrierfor insertion into the perfusion disposable. FIG. 3C is a schematic ofthe same assembled perfusion disposable embodiment shown in FIG. 3B,except that the ports on the cover assembly and the cutout (above theinserted chip for visualization, imaging, etc.) are now shown. FIG. 3Dis a schematic of the same perfusion disposable embodiment of FIG. 3C,but unassembled to show the relationships of the cover, reservoirs,skirt, chip and carrier.

FIG. 4A shows a side view of one embodiment of a chip carrier (with thechip inside) approaching (but not yet engaging) a side track of a skirtof one embodiment of the perfusion manifold assembly, the carrieraligned at an angle matching an angled front end portion of the sidetrack, the carrier comprising a retention mechanism configured as aupwardly protecting clip. Without being bound by theory, a suitablylarge angle permits chip engagement without smearing or prematureengagement of liquid droplets present on the chip and/or the perfusionmanifold assembly during the insertion and alignment processes. FIG. 4Bshows a side view of one embodiment of a chip carrier (with the chipinside) engaging a side track of a skirt of one embodiment of (but notyet linked to) the perfusion manifold assembly. FIG. 4C shows a sideview of one embodiment of a chip carrier (with the chip inside) fullyengaging a side track of a skirt of one embodiment of (but not yetlinked to) the perfusion manifold assembly (with an arrow showing thenecessary direction of movement to get a snap fit whereby the retentionmechanism will engage to prevent movement). FIG. 4D shows a side view ofone embodiment of a chip carrier (with the chip inside) detachablylinked to the perfusion manifold assembly, where the retention mechanismis engaged to prevent movement. While detachability and optionallyre-attachability is desirable in certain applications (for example,permitting chip removal to enable the addition of cells, imaging,performing various assays), in alternative embodiments, the linking isnot detachable. For example, an adhesive layer, glue and/or heat stakingmay be employed to provide a robust linkage that may pose a challenge indetachment or reattachment. FIG. 4E-1-4E-3 is a summary slideschematically showing one embodiment of a linking approach to theperfusion manifold comprising a 1) sliding action (4E-1), 2) pivoting(4E-2), and 3) snap fit (4E-3) so as to provide alignment and fluidicconnection in a single action. In the 1) sliding step, the chip (orother microfluidic device) is inserted into the carrier, which slidesalong to align the fluidic ports. In the 2) pivot step, the chip (orother microfluidic device) is pivoted until ports come into fluidcontact. In the 3) clip or snap fit step, the force needed to provide asecure seal is provided.

FIG. 5 is a schematic of one embodiment of a work flow (with arrowsshowing each progressive step), where the chip is linked (e.g. snappedin) to a disposable perfusion manifold assembly (“perfusiondisposable”), which in turn is positioned with other assemblies on aculture module, which is placed in an incubator. In alternativeembodiments, the culture module may comprise features of an incubator(e.g. a heat source and/or a source of warm moist air), so as to avoidthe need for a separate incubator. While the present inventioncontemplates “disposable” embodiments, the element may (alternatively)be reusable (e.g. as a cost consideration). In a further embodiment ofthe work flow or method, the chip can be placed in a carrier, thecarrier can be placed in a seeding guide (discussed and illustratedbelow), cells can be seeded into the chip, the carrier can be removedfrom the seeding guide, and the carrier can engage the perfusiondisposable (with the rest of the work flow as illustrated in FIG. 5).

FIG. 6 shows one embodiment of a removable tray with a plurality ofassemblies (with linked chips) positioned thereon, next to oneembodiment of a culture module with pressure points on a mating surfacethat correspond to the ports on the cover of each perfusion manifoldassembly held in the tray, such that they can be brought together by thetray mechanism so that pressure can be applied via the pressurecontrollers. The tray mechanism thereby attaches all of the perfusionmanifold assemblies to pressure or flow controllers in a single action(whether lifting the tray up or coming down to meet the tray), allowingfor a simultaneous linking.

FIG. 7 is a schematic of another embodiment of a culture module from theside, showing the platform for positioning the removable tray which ismoved upward into a mating surface so that pressure can be appliedthrough the pressure controllers (not shown).

FIG. 8A is a schematic of another embodiment showing the tray (or rack)and sub-tray (or nest) for transporting and inserting the perfusiondisposables (PDs) into the pressure module, which has a user interfaceon outside of the housing. FIG. 8B is a schematic of another embodimentshowing the trays (or racks) inserted within the housing of the culturemodule, which has a user interface. The illustrated nested design inwhich (in the present example) a tray carries multiple removablesub-trays provides the user with the flexibility to remove or carryvarious numbers of PDs depending on use. For example, the user may carrya full tray to a bio-safety cabinet in order to replenish media orcollect samples from all PDs in the tray, move a sub-tray of 3 PDs to amicroscope stage in order to image them without permitting the remainingPDs from dysregulating in term's of temperature or gas content, orremove or load a single PD for careful inspection or replacement.

FIG. 9A is a schematic of the interior of one embodiment of the pressuremodule (in an open position), showing the positioning of the tray (orrack), sub-tray (or nest), perfusion disposables (PDs) under a pressuremanifold (but not engaging it, so the clearance is sufficient to removethem), with the actuation assembly (including the pneumatic cylinder)above. Three microfluidic devices or perfusion disposables are shown toillustrate, although more (e.g. 6, 9 or 12) are typically used at once.

FIG. 9B is a schematic of the interior of one embodiment of the pressuremodule (in a closed position), showing the positioning of the tray (orrack), sub-tray (or nest), perfusion disposables (PDs) under thepressure manifold (and engaging it), with the actuation assembly(including the pneumatic cylinder) above. Again, three microfluidicdevices or perfusion disposables are shown to illustrate, although more(e.g. 6, 9 or 12) are typically used at once.

FIG. 10A is a schematic of one embodiment of the pressure manifold (50)showing the view of the PD engaging face (54) with several PD engaginglocations (in this case, six engaging locations). FIG. 10B shows amagnified portion of the engaging face (54) of the pressure manifold(50) highlighting the spring shuttle (55), valve seals (56) andalignment features (57) (so that the PD is aligned with the manifold).FIG. 10C is a schematic of another embodiment of the pressure manifold(50) showing the PD engaging face (54), along with an magnified portionhighlighting the lid compressor (58), valve seals (56) and alignmentfeatures (57) (so that the PD is aligned with the manifold). FIG. 10D isa schematic of one embodiment of the pressure manifold (50) showing thePD engaging face (54) from the side. FIG. 10E is a schematic of oneembodiment of the pressure manifold (50) showing the opposite face (67).FIG. 10F is a schematic of one embodiment of the pressure manifold (50)showing the PD engaging face (54) view with the PD guide (68) and lowerbacker plate (69) removed, highlighting one spring carrier (70) andspring (71) (out of many) by showing it removed from the manifold body,along with one seal (72), shuttle (73), and valve body (74) (out ofmany) by showing it removed from the manifold body. An exterior spring(75) adapted to depress the pressure manifold against the perfusiondisposables is also highlighted by showing it removed. FIG. 10G is aschematic of one embodiment of the pressure manifold (50) showing theopposite face (67) (not the PD engaging face) view with the upper backerplate (76) and capping strip (77) removed. Illustrated are manifoldrouting channels (78), which are adapted to direct and optionallydistribute pressure and/or fluid from one or more pressure ports.Additionally illustrated is one screw (79) (among many) and one gas port(80) (out of five, including both gas and vacuum ports) by showing itremoved from the manifold body (50). FIG. 10H is a schematic of oneembodiment of the pressure manifold (50) showing a top view of themanifold routing channels (78) and one port (81) among many.

FIG. 11A is a schematic of one embodiment of a valve (59) (a Schradervalve) in the pressure manifold (50), showing the silicone membrane(60), shuttle (61), air inlet (62) and cover plate (63). FIG. 11B is aside view and FIG. 11C is a top view photograph of one embodiment of avalve for the pressure manifold, showing the valve seat (64) and amembrane (60) acting as the valve seal. FIG. 11D is an interior sideview schematic of one embodiment of the pressure manifold (50) showing aplurality of valves (59) in the manifold body, the poppet (65), valveseal (66) and PD cover (11). In operation (to engage the PD), the valveseal (66) deflects with the displacement of the poppet (65).

FIG. 12A is a schematic of one embodiment of a connection schemecomprising a tube connecting manifold (82) permitting four culturemodules (30) (three are shown) to be connected inside a single incubator(31) using one or more hub modules (the two circles provide magnifiedviews of a first end (83) and second end (84) of the connections). FIG.12B is a photograph of gas hubs and vacuum hubs (collectively 85), alongwith the tubing (86) for the connection shown in FIG. 12A.

FIG. 13 is a photograph of one embodiment of an incubator (from theoutside with the outer door closed) containing shelves (not shown) whichcan support the perfusion manifold assemblies of the present invention.The incubator may have automated liquid handling, imaging and sensingfeatures for automatic experiments, evaluating cell viability and/orcollecting experimental results. In one embodiment, the microfluidicdevices are linked during incubation.

FIG. 14A-14B is a schematic showing one embodiment of connecting twomicrofluidic devices, resulting in the introduction of air bubbles intothe microchannels. FIG. 14A shows two fluidically primed devices (thefluid is shown with a meniscus) with ports and microchannels that arenot yet connected. FIG. 14B shows the devices of FIG. 14A contacting ina manner that results in the introduction of air bubbles (air is shownin the middle, between each meniscus) into the ports (and ultimately,the microchannels).

FIG. 15A-15B is a schematic showing one embodiment of connecting twomicrofluidic devices (or a microfluidic device to a fluid source)utilizing a drop-to-drop approach, resulting in no air bubbles. FIG. 15Ashows two fluidically primed devices with microchannels with protrudingdroplets formed on the surfaces of the devices but not in the areasaround the fluidic vias or port, and more particularly, formed directlyon and above the ports. FIG. 15B shows that when the surfaces come neareach other during a connection, the droplet surfaces join typicallywithout introducing any air bubbles.

FIG. 16A shows one embodiment for bringing a microfluidic device intocontact with a fluid source or another microfluidic device, wherein themicrofluidic device approaches from the side. FIG. 16B shows oneembodiment for bringing a microfluidic device into contact with a fluidsource or another microfluidic device, wherein the microfluidic deviceapproaches from the side and underneath, so as to cause a drop-to-dropconnection establishing fluidic communication (FIG. 16C). FIG. 16D showsyet another approach for brings a microfluidic device into contact witha fluid source or another microfluidic device, wherein the microfluidicdevice pivots.

FIG. 17 is a schematic showing a confined droplet (22) on the surface(21) of a microfluidic device (16) in the via or port.

FIG. 18 is a schematic showing a confined droplet (22) above the surface(21) of a microfluidic device (16) in the area of the via or port,wherein the droplet sits on a molded-in pedestal or mount (42) andcovers the mouth of the port and protrudes above the port, and where theport is in fluidic communication with a microchannel.

FIG. 19 is a schematic showing a confined droplet (22) above the surface(21) of a microfluidic device (16) in the area of the via or port,wherein the droplet sits on a gasket (43), covers the mouth of the port,and protrudes above the port, and where the port is in fluidiccommunication with a microchannel.

FIG. 20 is a schematic showing a confined droplet (22), a portion of thedroplet positioned below the surface (21) of a microfluidic device (16)in the area of the via or port, wherein the droplet sits on a molded-indepression or recess (44) and covers the mouth of the port, with aportion protruding above the surface, and where the port is in fluidiccommunication with a microchannel.

FIG. 21 is a schematic showing a confined droplet (22), a portion of thedroplet positioned below the surface (21) of a microfluidic device (16)in the area of the via or port, wherein the droplet sits in asurrounding gasket and covers the mouth of the port, with a portionprotruding above the gasket.

FIG. 22A-22B is a schematic showing a surface modification embodiment.FIG. 22A employs a hydrophilic adhesive layer or sticker (45) upon whichthe droplet (22) spreads out to the edges of the sticker, constrained bya surrounding hydrophobic surface. FIG. 22B shows a droplet (22)spreading out on a hydrophilic surface of the device, constrained by asurrounding hydrophobic surface.

FIG. 23 is a schematic showing a surface modification embodimentemploying surface treatment (indicated by downward projecting arrows) inconjunction with a mask (41).

FIG. 24A-24D is a schematic of one embodiment of a drop-to-dropconnection scheme whereby a combination of geometric shapes and surfacetreatments are used to control the droplet. FIG. 24A shows an embodimentof the microfluidic device or “chip” comprising a fluid channel andports, having an elevated region at each port (e.g. a pedestal orgasket). FIG. 24B shows the hydrophilic channel filled with fluid wherethe droplet radius is balanced at each end (i.e. at the port openings).FIG. 24C shows one portion of the microfluidic device of FIG. 24B withan upward projecting droplet (22) approaching (but not yet in contactwith) one portion of the mating surface of the perfusion manifoldassembly, which also has a projecting droplet (in this case, the droplet(23) is projecting downward). FIG. 24D shows the same portion of themicrofluidic device of FIG. 24C with the upward projecting droplet (22)of the microfluidic device making contact with (and merging with) thedownwardly projecting droplet (23) of the perfusion manifold assembly.

FIG. 25A-25B shows an embodiment of drop-to-drop connecting usingsurface treatments alone (i.e. without geometric shapes such aspedestals or gaskets). FIG. 25A shows an embodiment of the perfusionmanifold assembly comprising a fluid channel and a port. FIG. 25B showsthe hydrophilic channel filled with fluid to a level (e.g. height of thecolumn of fluid).

FIG. 26 is a chart showing (without being bound by theory) therelationship between the port diameter (in millimeters) and the maximumhydrostatic head (in millimeters) that the stabilized droplet cansupport.

FIG. 27 shows an embodiment where the microfluidic device (“chip”) islinked from below to the perfusion manifold assembly (above) at a portwith a venting gasket (43), where the assembly does not cover or closeoff the gasket, allowing any air trapped during the linking to be ventedout (right hand arrow). It may be desirable to ensure that any airpreferentially flows out through the venting gasket rather than continueto flow through the channels. In some embodiments, this preferentialflow is encouraged by subjecting fluid in the fluid channel of theassembly (left hand arrow) to a first pressure (P1) and fluid in themicrofluidic device channel to a second pressure (P2), where P1 and P2are greater than the back-pressure of the venting gasket. In someembodiments, the pressure P1 and/or P2 are applied using a pressuresource and/or gravitational head. In some embodiments, the pressure P1and/or P2 are generated by the flow resistance of the fluid.

FIG. 28 shows another embodiment where the microfluidic device (16)(“chip”) is linked from below to the perfusion manifold assembly (10)(above) at a port with a venting gasket (43), where the assembly coversthe gasket (i.e. the gasket is enclosed by the assembly mating surface),but where there is a path in the assembly above the gasket to allow anyair trapped during the linking to be vented out.

FIG. 29 shows another embodiment where the microfluidic device (16)(“chip”) is linked from below to the perfusion manifold assembly (10)(above) at a port with a venting gasket (43), where the fluid path goesover the gasket (the gasket can be larger if desired). This embodimentfacilitates the removal of air trapped during the linking includingsmaller bubbles, since, without being bound by theory, it enablessmaller bubbles to interact with (“wet”) the venting gasket.

FIGS. 30A-30B and 31A-31B are a series of still photos from a videoshowing one embodiment of the microfluidic device (having dropletsprotruding from gaskets) moving along a essentially linear (i.e. alongthe x axis in the x/y plane) rail or guide track of a fluid source, ormicrofluidic device such as the perfusion manifold assembly (compareFIG. 30A to 30B) until it gets close (FIG. 31A) to the correspondingports of the perfusion manifold assembly, whereupon a combination ofmovement in the x axis and z axis (i.e. side movement and upwardmovement) causes the droplets to merge and the chip to link (FIG. 31B).

FIG. 32A shows one embodiment of a fluidic backplane comprisingserpentine fluid resistor channels (91), vacuum channels (92) and outputchannels (93). FIG. 32B is an edge view. FIG. 32C shows the chipengagement bosses (94) of the fluidic backplane, which serve as itsfluidic outlet ports, along with assembly alignment features (95) and avisualization cutout (96) which permits microscopy and other imaging.

FIG. 33A-33B shows a schematic of an illustrative microfluidic device or“organ-on-chip” device. The assembled device is schematically shown inFIG. 33A. FIG. 33B shows an exploded view of the device of FIG. 33A.

FIG. 34 is a schematic showing an embodiment with two membranes.

FIG. 35A shows first and second end caps (106 and 107) and first andsecond side panels (108 and 109) as the components of one embodiment ofan unassembled culture stand or holder (100). FIG. 35B shows the chip(16) and carrier (17) within a seeding guide, the seeding guideapproaching (but not engaging) the stand (100). FIG. 35C shows sixseeding guides comprising carriers (17) (with chips) mounted on thestand (100).

FIGS. 36A-C are photographs of a perfusion manifold assembly embodimentthat lacks a skirt (or other projection) with side tracks for engaging achip (or other microfluidic device) in a carrier). Instead, the base(110) of the assembly (10) is configured to accept the carrier (17) fromunderneath in a Lego™ block type connection (instead of from the side),i.e. the base (110) has a cavity (111) and openings (112) dimensioned toaccept the carrier (17), while the carrier's handle or tab (18) isconfigured to fit in the openings (112). FIG. 36A is a topside view ofthe assembly (10) before engaging the carrier (17) and chip (16). FIG.36B shows an underside view of the assembly (10) with fluidic outletports (94) configured to align with ports (2) on the chip (16). FIG. 36Cshows the assembly (10) engaged with the carrier such that the carriertab (18) is positioned in the openings (112).

DEFINITIONS

“Bond number” is a dimensionless ratio of gravity forces to capillaryforces on a liquid interface. When the Bond number is high air, liquidinterfaces tend to be shaped by gravity. When the Bond number is low,those surfaces tend to be shaped by the capillary force.

“Hydrophobic reagents” are used to make “hydrophobic coatings” onsurfaces (or portions thereof), including projections, platforms orpedestals at or near ports, as well as mating surfaces (or portionsthereof). It is not intended that the present invention be limited toparticular hydrophobic reagents. In one embodiment, the presentinvention contemplates the use of silanes to make hydrophobic coatings,including but not limited to halogenated silanes and alkylsilanes. Inthis regard, it is not intended that the present invention be limited toparticular silanes; the selection of the silane is only limited in afunctional sense, i.e. that it render the surface hydrophobic. Thepresent invention also contemplates using commercially availableproducts, such as the Rain-X™ product which is a synthetic hydrophobicsurface-applied product that causes water to bead, most commonly used onglass automobile surfaces.

A surface or a region on a surface is “hydrophobic” when it displays(e.g. advancing) contact angles for water greater than approximatelyninety (90) degrees (in many cases, it is preferable that both advancingand receding contact angles are greater than approximately 90 degrees).In one embodiment, the hydrophobic surfaces of the present inventiondisplay advancing contact angles for water between approximately ninety(90) and approximately one hundred and ten (110) degrees. In anotherembodiment, hydrophobic surfaces have regions displaying advancingcontact angles for water greater than approximately one hundred and ten(110) degrees. In another embodiment, hydrophobic surfaces have regionsdisplaying receding contact angles for water greater than approximately100 degrees. It is important to note that some liquids, and particularlysome biological liquids, contain elements that may coat a surface afterwetting it, thereby affecting its hydrophobicity. In the context of thepresent invention, it may be important that a surface resists suchcoating from a liquid of intended use, for example, that such coatingdoes not create an advancing and/or receding contact angle that is lessthan 90 degrees over the duration that the surface remains wetted by thesaid liquid.

A surface or a region on a surface is “hydrophilic” when it displays(e.g. advancing) contact angles for water less than approximately ninety(90) degrees, and more commonly less than approximately seventy (70)degrees (in many cases it is preferable that both the advancing andreceding contact angles are less than approximately 90 degrees orapproximately 70 degrees).

Measured contact angles can fall in a range, i.e. from the so-calledadvancing (maximal) contact angle to the receding (minimal) contactangle. The equilibrium contact is within those values, and can becalculated from them.

Hydrophobic surfaces “resist wetting” by aqueous liquids. A material issaid to resist wetting by a first liquid where the contact angle formedby the first liquid on the material is greater than 90 degrees. Surfacescan resist wetting by aqueous liquids and non-aqueous liquids, such asoils and fluorinated liquids. Some surfaces can resist wetting by bothaqueous liquids and non-aqueous liquids. Hydrophobic behavior isgenerally observed by surfaces with critical surface tensions less than35 dynes/cm. At first, the decrease in critical surface tension isassociated with oleophilic behavior, i.e., the wetting of the surfacesby hydrocarbon oils. As the critical surface tensions decrease below 20dynes/cm, the surfaces resist wetting by hydrocarbon oils and areconsidered oleophobic as well as hydrophobic.

Hydrophilic surfaces “promote wetting” by aqueous liquids. A material issaid to promote wetting by a first liquid where the contact angle formedby the first liquid on the material is less than 90 degrees, and morecommonly less than 70 degrees.

As used herein, the phrases “linked,” “connected to,” “coupled to,” “incontact with” and “in communication with” refer to any form ofinteraction between two or more entities, including mechanical,electrical, magnetic, electromagnetic, fluidic, and thermal interaction.For example, in one embodiment, channels in a microfluidic device are influidic communication with cells and (optionally) a fluid reservoir. Twocomponents may be coupled to each other even though they are not indirect contact with each other. For example, two components may becoupled to each other through an intermediate component (e.g. tubing orother conduit).

“Channels” are pathways (whether straight, curved, single, multiple, ina network, etc.) through a medium (e.g., silicon, plastic, etc.) thatallow for movement of liquids and gasses. Channels thus can connectother components, i.e., keep components “in communication” and moreparticularly, “in fluidic communication” and still more particularly,“in liquid communication.” Such components include, but are not limitedto, liquid-intake ports and gas vents.

“Microchannels” are channels with dimensions less than 1 millimeter andgreater than 1 micron. Additionally, the term “microfluidic” as usedherein relates to components where moving fluid is constrained in ordirected through one or more channels wherein one or more dimensions are1 mm or smaller (microscale). Microfluidic channels may be larger thanmicroscale in one or more directions, though the channel(s) will be onthe microscale in at least one direction. In some instances the geometryof a microfluidic channel may be configured to control the fluid flowrate through the channel (e.g. increase channel height to reduce shear).Microfluidic channels can be formed of various geometries to facilitatea wide range of flow rates through the channels.

The present invention contemplates a variety of “microfluidic devices,”including but not limited to microfluidic chips (such as that shown inFIG. 3A), perfusion manifold assemblies (without chips), and perfusionmanifold assemblies engaged with microfluidic chips (such as that shownin FIG. 3B). However, the methods described herein for engagingmicrofluidic devices (e.g. by drop-to-drop connections), and forperfusing microfluidic devices are not limited to the particularembodiments of microfluidic devices described herein, and may be appliedgenerally to microfluidic devices, e.g. devices having one or moremicrochannels and ports.

A “stable droplet” is a droplet of media that does not experiencesignificant movement away from its intended location (e.g. to remain incontact with a fluidic port) and preferably does not experience asignificant (>10%) change in volume or placement on a microfluidicdevice over the course of several seconds, and more preferably oneminute, and even more preferably several minutes (2-10 minutes). In apreferred embodiment, the present invention contemplates a stabledroplet during drop-to-drop engagement. A surface may intrinsically(e.g. because of what it is made of) be able to stably retain, or bemade to stably retain, a droplet, meaning that the droplet will notspontaneously expand or shift beyond a limited (or designated) area.Stable droplets do not experience a significant change in volume orplacement. The present invention contemplates this spatial control ofdroplets, i.e. retaining the droplet within a defined spatial extentand/or retaining the droplet within the spatial extent of the one ormore regions. In a preferred embodiment, the present inventioncontemplates both preventing the droplet from extending too far, andensuring that it is centered on the port (i.e. making sure that the arearight on top of the fluidic port remains covered by the droplet). Interms of preventing the droplet from extending or spreading too wide,the present invention contemplates, in one embodiment, retaining thedroplet within the spatial extent of the one or more regions. In aparticularly preferred embodiment, the present invention contemplatespreventing the droplet from shifting away during manipulation (i.e.rolling away on the surface as the microfluidic device or chip is movedaround or even inverted. Of course, such movements are contemplatedwithout violent shaking. A droplet that is found to be stable if aparticular engagement procedure is used, may be found unstable ifanother procedure (e.g. more violent procedure) is utilized.

“Controlled engagement” refers to engagement of two devices that allowsfor both adequate alignment of vias or ports, and smooth drop-to-dropconnection, which does not result in loss of droplet stability. If thedevices, for example, snap violently into place or the droplets onopposite devices touch prior to engagement, droplet stability will becompromised.

General Description of the Invention

In one embodiment, the present invention contemplates a perfusionmanifold assembly that allows for perfusion of a microfluidic device,such as an organ on a chip microfluidic device comprising cells thatmimic cells in an organ in the body or at least one function of anorgan, that is (preferably detachably) linked with said assembly so thatfluid enters ports of the microfluidic device from a fluid reservoir,optionally without tubing, at a controllable flow rate. In oneembodiment (as shown in FIGS. 1A, 1B and 1C), the perfusion manifoldassembly (10) comprises i) a cover or lid (11) configured to serve as totop of ii) one or more fluid reservoirs (12), iii) a capping layer (13)under said fluid reservoir(s), iv) a fluidic backplane (14) under, andin fluidic communication with, said fluid reservoir(s), said fluidicbackplane comprising a fluidic resistor, and v) a projecting member orskirt (15) for engaging the microfluidic device (16) or chip which ispreferably positioned in a carrier (17), the chip having one or moremicrochannels (1) and in fluidic communication with one or more ports(2). The assembly can be used with or without the lid or cover. Otherembodiments (discussed below) lack a skirt or projecting member. In oneembodiment, the carrier (17) has a tab or other gripping platform (18),a retention mechanism such as a clip (19), and a visualization cutout(20) for imaging the chip. The cutout (20) can enable placing a carrier(e.g. a carrier engaged with the perfusion manifold assembly or “pod” ornot so engaged) onto a microscope or other inspection device, allowingthe chips to be observed without having to remove the chip from thecarrier. In one embodiment, the fluidic resistor comprises a series ofswitchbacks or serpentine fluid channels. FIG. 32 shows an enhancedschematic of one embodiment of the backplane, showing the fluid resistorchannels (32A) and chip engagement bosses (32C) or ports. A variety offluid resistors designs are contemplated, as described more fully inU.S. Provisional Application Ser. Nos. 62/024,361 and 62/127,438, whichbecame PCT/US2015/040026, hereby incorporated by reference (and inparticular, the discussion of resistors, resistor design, and pressuresis incorporated herein by reference). In one embodiment, the perfusionmanifold assembly is made of plastic and is disposable, i.e. it isdisposed of after docking with and perfusing a microfluidic device.While the present invention contemplates “disposable” embodiments, theelement may (alternatively) be reusable (e.g. as a cost consideration).

In one embodiment, the microfluidic device (e.g. chip) (16) may first beplaced in a carrier (17) (e.g. chip carrier) before engaging theperfusion manifold assembly (10) or may engage the assembly directly. Ineither case, the (optional) detachable linking of the microfluidicdevice with the manifold should either a) prevent air from entering themicrochannels, or b) provide a way for undesirable air to be removed orvented out of the system. Indeed, air removal may be needed in someembodiments during both chip attachment and use of the microfluidicdevice.

In one embodiment for preventing air from entering the microchannels,the microfluidic device is detachably linked using a “drop-to-drop”“chip-to-cartridge” connection. In this embodiment, an inlet port of themicrofluidic device has a droplet (22) projecting therefrom (FIG. 15A),and the surface of the perfusion manifold assembly or “cartridge” (10)for engaging the device has a corresponding droplet (23). When the twoare brought together (FIG. 15B), the droplets merge allowing for fluidiccommunication without the introduction of air into the channels. In oneembodiment, the chip carrier is designed so as to not interfere with the“drop-to-drop” connection. For example, the carrier, in one embodiment,surrounds the sides, but not the mating surface (21) of the microfluidicdevice. It should be noted that FIG. 15A shows a skirt-free perfusionmanifold (10) where the microfluidic device or chip engages fromunderneath (rather than from the side).

It is not intended that the present invention be limited to only onemanner for detachably linking the microfluidic device. In oneembodiment, the microfluidic device, such as an organ on a chipmicrofluidic device comprising cells that mimic one or more functions ofcells in an organ in the body or at least one function of an organ,approaches the assembly from the side (FIG. 16A) or underneath (FIG.16B), with the droplet (22) projecting upward, while the correspondingdroplet (23) on the assembly (or other type of fluid source) projectsdownward. The microfluidic device (or the device carrier) may comprisesa portion (24) configured to engage a side track (25) or other guidemechanism. In another embodiment, the microfluidic device, such as anorgan on a chip microfluidic device comprising cells that mimic cells inan organ in the body or at least one function of an organ, approachesthe assembly from above, with the droplet projecting downward, while thecorresponding droplet on the assembly projects upward. In still anotherembodiment, the microfluidic device, such as an organ on a chipmicrofluidic device comprising cells that mimic cells in an organ in thebody or at least one function of an organ, approaches the assembly fromthe side and is positioned by pivoting (FIG. 16D, see the arrow) about ahinge, socket, or other pivot point (26). In still another embodiment,the microfluidic device engages in the manner of an audio cassette or CDwith the droplet projecting upward, while the corresponding droplet onthe assembly projects downward, where there is a combined sidewaysmovement and upward movement (FIGS. 16B-16C).

In one embodiment, the microfluidic device (16) is detachably linkedwith the perfusion manifold assembly (10) by a clipping mechanism thattemporarily “locks” the microfluidic device, including organ-on-chipdevices, in place (FIGS. 4A, 4B, 4C and 4D). In one embodiment, theclipping or “snap fitting” involves a projection on the carrier (19)which serves as a retention mechanism when the microfluidic device (16)is positioned. In one embodiment, the clipping mechanism is similar tothe interlocking plastic design of a Lego™ chip and comprises astraight-down clip, friction fit, radial-compression fit or combinationthereof. However, in another embodiment, the clipping mechanism istriggered only after the microfluidic device, or more preferably, thecarrier (17) comprising the microfluidic device (16), engages theperfusion manifold assembly (or cartridge) on a guide rail, side slot,internal or external track (25) or other mechanism that provides astable glide path for the device as it is conveyed (e.g. by machine orby hand) into position. The guide rail, side slot, internal or externaltrack (25) or other mechanism can be, but need not be, strictly linearand can be positioned in a projecting member or skirt (15) attached tothe main body of the perfusion manifold assembly (10). In oneembodiment, the beginning portion of the guide rail (25) (or side slot,internal or external track or other mechanism) comprises an angled slide(27) which provides a larger opening for easier initial positioning,followed by a linear or essentially linear portion (28). In oneembodiment, the end portion (29) (close to the corresponding ports ofthe assembly) of an otherwise linear (or essentially linear) guide rail(25) (or side slot, internal track or other mechanism) is angled (orcurves) upward (FIG. 16B) so that there is a combination of linearmovement (e.g. initially) and upward movement to achieve linking.

In several embodiments, it is important that droplets remain placed attheir corresponding fluidic port despite the motion of their substrateor any period of upside-down orientation. In addition, it is desirablethat the droplets retain their size, for example, so that thedrop-to-drop process is consistent regardless of the speed of theengagement process. Accordingly, the present invention contemplatesdesigns and method to provide stable droplets. Stable droplets arecontemplated for aqueous as well as non-aqueous liquids. Although wefocus our examples without loss of generality on aqueous droplets, onefamiliar with the art should be able to adapt the examples andparticularly the use of hydrophilic and hydrophobic regions or materialsbased on the wetting properties of the liquid. In some embodiments, adroplet may be restricted within a first region of a substrate bysurrounding the first region with a second region, wherein the secondregion is hydrophobic (or more generally, with a propensity againstwetting by the droplet's liquid). The said second region may behydrophobic due to selection of one or more hydrophobic materials thatit comprises (e.g. PTFE, FEP, certain grades of Nylon, etc.), surfacetreatment (e.g. plasma treatment, chemical treatment, ink treatment),the use of a gasket (e.g. a film, an o-ring, an adhesive gasket), bymasking during treatment of at least one other region of the substrate,or a combination thereof. In some embodiments, a droplet may berestricted within a first region of a substrate by surrounding the firstregion with a geometric feature. In some embodiments, the geometricfeature may be a ridge or a depression. Without being bound by theory,such features may act to restrict the droplet by means of their edges,which interact with the surface layer of the droplet (andcorrespondingly with the surface tension of the droplet), for example,by “pinning” the surface of the droplet. In some embodiments, a dropletmay be restricted to cover a first region of a substrate by adapting thefirst region to be hydrophobic (or more generally, with a propensity forwetting by the droplet's liquid). The said first region may behydrophilic due to selection of one or more hydrophilic materials thatis comprises (e.g. PMMA, PLA), surface treatment (e.g. plasma treatment,chemical treatment, ink treatment), the use of a gasket (e.g. a film, ano-ring, an adhesive gasket), by masking during the treatment of at leastone of other region of the substrate, or a combination thereof.

In one embodiment, the mating surface (21) of a microfluidic device (orat least a portion thereof adjacent the port opening) is hydrophobic ormade hydrophobic (or protected with a mask during plasma treatment tokeep it from becoming hydrophilic). In one embodiment, the matingsurface of a perfusion manifold assembly or cartridge (or at least aportion thereof adjacent the port opening) is hydrophobic or madehydrophobic (or protected with a mask during plasma treatment to keep itfrom becoming hydrophilic). In one embodiment, both the mating surfaceof the microfluidic device (or at least a portion thereof adjacent theport opening) and the mating surface of the perfusion manifold (or atleast a portion thereof adjacent the port opening) is hydrophobic ormade hydrophobic (or protected with a mask during plasma treatment tokeep it from becoming hydrophilic).

The advantage of the carrier is that the surfaces of the microfluidicdevice need not be touched during the detachable linkage with theperfusion manifold assembly. The carrier can have a plate, platform,handle or other mechanism for gripping the carrier (18), withoutcontacting the mating surface (21) of the microfluidic device (16). Theretention mechanism (19) can comprise a projection, hook, latch or lipthat engages one or more portions of the perfusion manifold assembly,and more preferably the skirt of the perfusion manifold assembly, toprovide a “snap fit.”

In other embodiments (FIGS. 27, 28 and 29), one or more gaskets can beused to vent air (e.g. any air that has been introduced because of thedetachable linking of the microfluidic device with the perfusionmanifold assembly). While in one embodiment, bubbles can be trapped (andtheir impact thereby limited), in an alternative embodiment, they arevented. One method involves use of hydrophobic vent material (molded orsheet). For example, the hydrophobic vent material may comprise PTFE,PVDF, hydrophobic grades of Nylon, or a combination thereof. In someembodiments venting can be accomplished by employing materials thatdisplay high gas permeability (e.g. PDMS). In other embodiments, ventingcan be accomplished by employing porous materials, for example, sinteredmaterials, porous membranes (e.g. track-etched membranes, fiber-basedmembranes), open-cell foams, or a combination thereof. In a preferredapproach, air escapes from a vented (or venting) gasket. In someembodiments, the perfusion manifold assembly or microfluidic devicecomprise a vent adapted to provide a path for undesired gas to escape.

Once a microfluidic device (or “chip”) has docked with the perfusionmanifold assembly, the assembly-chip combination can be placed into anincubator (31) (typically set at a temperature above room temperature,e.g. 37° C.), or more preferably, into a culture module (30) capable ofholding a plurality of assembly-chip combinations, the culture moduleconfigured to fit on an incubator shelf (see FIG. 5). This allows forthe easy handling of many (e.g. 5, 10, 20, 30, 40, 50 or more)microfluidic devices at one time. For example, where the culture modulecomprises 9 assembly-chip combinations, and an incubator is sized for 6to 9 culture modules, between 54 and 81 “organs-on-chip” can be handledin a single incubator (FIG. 5 and FIG. 8). In another example, where theculture module comprises 12 assembly-chip combinations, and an incubatoris sized for 4 to 6 culture modules, between 48 and 72 “organs-on-chip”can be handled in a single incubator. The perfusion manifold can beeasily removed and inserted into the culture module without breaking thefluidic connections to the chip. In one embodiment, the culture moduleis capable of maintaining the temperature above room temperature, e.g.37° C., without being placed in an incubator.

The culture module (30), in one embodiment (FIG. 6), comprises aremovable tray (32) for positioning the assembly-chip combinations, apressure surface (33), and pressure controllers (34), along with anoptional user interface (46) to control the movement of the variouselements. In one embodiment, the tray (32) can slide. In one embodiment,the tray is positioned on the culture module and the tray is moved upvia a tray mechanism (35) to engage the pressure surface (33) of theculture module, i.e. the cover or lid (11) of the perfusion manifoldassembly (10) engages the pressure surface of the culture module (30).Multiple perfusion assemblies (10) can be attached to the pressurecontrollers in a single action by the tray mechanism. In anotherembodiment, the tray is positioned on the culture module and thepressure surface of the culture module (30) is moved down to engage thetray (32), i.e. the cover or lid (11) of the perfusion manifold assembly(10). In either case, in one embodiment (FIGS. 2A and 2B), the cover orlid comprises ports such as through-hole ports (36) that are engaged bycorresponding pressure points on the pressure surface (33) of theculture module. These ports (36), when engaged, transmit appliedpressure inward through the cover and through a gasket (37) and applythe pressure to the fluid in the reservoirs (12) of the perfusionmanifold assembly (10). Thus, in this embodiment, pressure is appliedthrough the lid (11) and the lid seals against the reservoir(s). Forexample, when on applies 1 kPa, this nominal pressure results, in oneembodiment, in a flow rate of approximately 30-40 uL/hr. Alternatively,these ports (36), when engaged, move inward on the cover so as tocontact the gaskets (i.e. the ports act essentially like plungers).

FIG. 8A is a schematic of another embodiment of the culture module (30)showing the tray (or rack) (32) and sub-tray (or nest) for transportingand inserting the perfusion disposables (10) into the culture module,which has two openings (48, 49) in the housing to receive the trays, anda user interface (46) to control the process of engaging the perfusiondisposables and applying pressure. A typical incubator (not shown) canhold up to six modules (30). FIG. 8B is a schematic of the sameembodiment of FIG. 8A, but showing both of the trays (or racks) (32)inserted into the two openings (48, 49) in the housing (53) of thepressure module (30), which has a user interface (46) (e.g. LCD screen)to control the process.

FIG. 9A is a schematic of the interior of one embodiment of the module(i.e. the housing has been removed), showing the pressure manifold (50)in an open position, with the positioning of the tray or rack (32),sub-tray or nest (47), perfusion disposables (10) under the pressuremanifold (50) but not engaging it (so the clearance is sufficient toremove them), with the actuation assembly (51) including the pneumaticcylinder (52) above.

FIG. 9B is a schematic of the interior of one embodiment of the module(i.e. the housing has been removed), showing the pressure manifold (50)in a closed position, with the positioning of the tray or rack (32),sub-tray or nest (47), perfusion disposables (10) under the pressuremanifold (50) and engaging it, with the actuation assembly (51)including the pneumatic cylinder (52) above. The pressure manifold (50)simultaneously engages all of the perfusion disposables (10) while mediaperfusion is required or needed. Independent control of the flow rate inthe top and bottom channels of the chip (16) can be achieved. Thepressure manifold (50) can disengage (without complicated fluiddisconnects) as desired to allow removal of the trays (32) or nests (47)for imaging or other tasks. In one embodiment, the pressure manifold(50) can simultaneously disengage from a plurality of perfusion manifoldassemblies. In one embodiment, the perfusion disposables (10) are notrigidly fixed inside the nests (47), allowing them to locate relative tothe pressure manifold (50) as it closes. In a preferred embodiment,integrated alignment features in the pressure manifold (50) provideguidance for each perfusion disposable (10).

In one embodiment, the cover or lid is made of polycarbonate. In oneembodiment, each through-hole port is associated with a filter (38)(e.g. a 0.2 um filter). In one embodiment, the filters are aligned withholes (39) in a gasket positioned underneath the cover.

A culture module comprising a pressure manifold is contemplated thatallows the perfusion and optionally mechanical actuation of one or moremicrofluidic devices, such as organ-on-a-chip microfluidic devicescomprising cells that mimic at least one function of an organ in thebody. FIG. 10A is a schematic of one embodiment of the pressure manifold(50) showing the view of the PD engaging face (54) with several PDengaging locations (in this case, six engaging locations). FIG. 10Bshows a magnified portion of the engaging face (54) of the pressuremanifold (50) highlighting the spring shuttle (55), valve seals (56) andalignment features (57) (so that the PD is aligned with the manifold).The spring shuttle is an optional means by which the pressure manifoldmay sense the presence of a PD in the particular PD engaging location.In a specific embodiment, the presence of a PD depresses the springshuttle, which opens one or more valves disposed within the pressuremanifold to enable the application of pressure or fluid flow to the PD.In turn, when a PD is absent, the shuttle is not depressed, leaving thevalve closed; this is intended to prevent pressure or fluid leakage. Theillustrated valve seals are adapted to form pressure and/or fluid sealsagainst corresponding features in the PD and if present, a pressure lid.FIG. 10C is a schematic of another embodiment of the pressure manifold(50) showing the PD engaging face (54), along with an magnified portionhighlighting the lid compressor (58), valve seals (56) and alignmentfeatures (57) (so that the PD is aligned with the manifold). Lidcompressors may apply force onto a pressure lid in order to aid theestablishment of maintenance of a pressure and/or fluidic seal betweenthe pressure lid and reservoirs. In one embodiment, the lid compressorscomprise springs, elastomeric material, pneumatic actuators orcombination thereof, which can be selected and sized to apply a forcecorresponding to the force required to maintain the said pressure and/orfluidic seal. FIG. 10D is a schematic of one embodiment of the pressuremanifold (50) showing the PD engaging face (54) from the side. FIG. 10Eis a schematic of one embodiment of the pressure manifold (50) showingthe opposite face (67). FIG. 10F is a schematic of one embodiment of thepressure manifold (50) showing the PD engaging face (54) view with thePD guide (68) and lower backer plate (69) removed, highlighting onespring carrier (70) and spring (71) (out of many) by showing it removedfrom the manifold body, along with one seal (72), shuttle (73), andvalve body (74) (out of many) by showing it removed from the manifoldbody. An exterior spring (75) adapted to depress the pressure manifoldagainst the perfusion disposables is also highlighted by showing itremoved. FIG. 10G is a schematic of one embodiment of the pressuremanifold (50) showing the opposite face (67) (not the PD engaging face)view with the upper backer plate (76) and capping strip (77) removed.Illustrated are manifold routing channels (78), which are adapted todirect and optionally distribute pressure and/or fluid from one or morepressure ports. Additionally illustrated is one screw (79) (among many)and one gas port (80) (out of five, including both gas and vacuum ports)by showing it removed from the manifold body (50). FIG. 10H is aschematic of one embodiment of the pressure manifold (50) showing a topview of the manifold routing channels (78) and one port (81) among many.The routing channels can be produced using a number of methods known inthe art, including molding, machining, ablation, lamination, 3Dprinting, photolithography and a combination thereof.

FIG. 11A is a schematic of one embodiment of a valve (59) (a Schradervalve) in the pressure manifold (50), showing the silicone membrane(60), shuttle (61), air inlet (62) and cover plate (63). In thisembodiment, the spring shuttle is integrated into the valve and isadapted to depress the Schrader valve's poppet to actuate the valve.FIG. 11B is a side view and FIG. 11C is a top view photograph of oneembodiment of a valve for the pressure manifold, showing the valve seat(64) and a membrane (60) acting as the valve seal. FIG. 11D is aninterior side view schematic of one embodiment of the pressure manifold(50) showing a plurality of valves (59) in the manifold body, the poppet(65), valve seal (66) and PD cover (11). In operation (to engage thePD), the valve seal (66) deflects with the displacement of the poppet(65).

FIG. 12A is a schematic of one embodiment of a connection schemecomprising a tube connecting manifold (82) permitting four culturemodules (30) (three are shown) to be connected inside a single incubator(31) using one or more hub modules (the two circles provide magnifiedviews of a first end (83) and second end (84) of the connections). FIG.12B is a photograph of gas hubs and vacuum hubs (collectively 85), alongwith the tubing (86) for the connection shown in FIG. 12A. While thisconnection scheme is optional, it provides a convenient way to utilizemultiple culture modules with a single incubator.

DETAILED DESCRIPTION OF THE INVENTION

A. Pressure Lid

The present invention contemplates in one embodiment “perfusion manifoldassemblies” or “perfusion disposables,” which facilitate the culture ofOrgans-on-Chips within a culture instrument. While the present inventioncontemplates “disposable” embodiments, the element may (alternatively)be reusable (e.g. as a cost consideration).

In one embodiment, these perfusion disposables (PDs) include one or moreinput and one or more output reservoirs, as well as elements requiredfor pumping. In particular, in our present embodiment perfusiondisposables include one or more resistors (see FIG. 32A), which are usedfor pressure-driven pumping. In the pressure-driven embodiment, theinstrument creates or controls fluid flow by applying a pneumaticpressure (whether positive or negative) to one or more of thereservoirs. One advantage of this approach is that the pressure-drivendesign can avoid liquid contact with the instrument, which offersbenefits in terms of sterility and ease of use (e.g. avoiding gasbubbles in liquid lines). In some embodiments, the instrument appliespressure directly to the one or more reservoirs (with no lid). Asufficient pressure seal may be attained by integrated one or moregaskets on the perfusions disposable and/or the instrument (for example,as part of a pressure manifold). However, it is desirable that when theperfusion disposables are outside of the instrument the reservoirs areprotected from contamination, for example, from environmental particlesor airborne microbes. Accordingly, in the same embodiments it may bedesirable to provide a lid that a user can employ to cover thereservoirs when outside of the instrument and/or to employ PDembodiments that comprise a substrate that conveys pressure but blockscontamination (for example, a suitable filter disposed on a reservoir'sopening). However, such solutions typically pose drawbacks. Inparticular, expecting a user to place a lid requires the user to managelids while the perfusion disposables are engaged with the instrument andideally place the lids as soon as the PDs leave the instrument; in mostcircumstances, these actions adversely affect user experience. In turn,a filter disposed on a reservoir's opening typically blocks access tothe said reservoir by pipettes and other typical lab tools, therebyadversely limiting their ease of use.

According to an aspect of the present invention, we disclose a “pressurelid”, a lid that may be disposed on a microfluidic device or a deviceadapted to accept a microfluidic device (e.g. a perfusion disposable)even while the said device is engaged with an instrument, with thepressure lid adapted to permit the communication of pressure between theinstrument and the said device. The present invention contemplates thatin some embodiments, a pressure lid is a removable cover adapted to bedisposed onto one or more reservoirs of a microfluidic device or adevice adapted to accept a microfluidic device (e.g. a perfusiondisposable), the pressure lid comprising at least oneinstrument-interface port and at least one reservoir-interface port,wherein the pressure lid is adapted to convey pressure between at leastsome of the instrument-facing port and at least some of thereservoir-facing ports. In some embodiments, the pressure lid comprisesat least one “through hole” port—an opening that connect a first andsecond surface of the lid, wherein the opening on the first surface isadapted to form an instrument-facing port and the opening on the secondsurface is adapted to form a reservoir-facing port. In some embodiments,the though-hole port is round, rectangular, triangular, polygonal,rectilinear, curvilinear, elliptical, and/or curved. In someembodiments, however, the lid comprises a channel that links at leastone instrument-facing port and at least one reservoir-facing ports,which may not be disposed directly opposite each other. Such embodimentsmay be useful, for example, where there is a need to adapt betweenlocations of instrument interface and reservoir locations, for example,when it is desired for the same instrument to support the actuation of aplurality of versions of perfusion disposables.

In some embodiments, the pressure lid is adapted to form a pressure sealbetween said pressure lid and at least one reservoir. In someembodiments, the pressure lid is engaged with at least one reservoirforming a lid-to-reservoir pressure seal. In some embodiments, thepressure lid is adapted to faun a pressure seal between said pressurelid and at least one instrument. In some embodiments, the pressure lidis engaged with at least one instrument forming a lid-to-instrumentpressure seal. Any of the lid-to-reservoir seals and lid-to-instrumentseals may employ any sealing methodology known in the art and can beselected for example, from the list of face seal, radial seal, taperedseal, friction fit or a combination thereof. Any of the said seals mayemploy one or more gaskets, O-Rings, elastic materials, pliablematerials, adhesive, sealants, greases or combination thereof. It is notintended that the present invention be limited to a design that has aperfect pressure seal, as this may not be required. Rather, some amountof gas leakage can be tolerated, since the instrument may activelyregulate pressure, thereby compensating for the leak. The relaxing of arequirement to obtain a perfect seal on one or both sides can simplifydesign and reduce costs.

In some embodiments, the pressure lid comprises a load concentrator. Forexample, in some embodiments, the pressure lid comprises a ridgesurrounding at least one instrument-facing port. In some embodiments,the pressure lid comprises a ridge surrounding at least onereservoir-facing port. It is known in the art that such loadconcentrators can act to improve pressure seals by enhancing reliabilityor reducing the required force; designs known in the art include, forexample, rectangular, semi-circular, triangular, trapezoidal andpolygonal ridges. Accordingly, a load concentrator surrounding aninstrument-facing port may be employed to improve a lid-to-instrumentpressure seal, and a load concentrator surrounding a reservoir-facingport may be used to improve a lid-to-reservoir pressure seal.

In some embodiments, the pressure lid comprises a filter. For example,the pressure lid may comprise a membrane filter, sintered filter,fiber-based filter and/or track-etched filter. In some embodiments, thesaid filter is disposed within or abutting a through-hole port and/orone of its openings. In some embodiments, the said filter is disposedwithin or abutting a channel included in the lid and/or one of theopenings of said channel.

In some embodiments, the filter is selected to improve the sterility ofa reservoir and/or block particles, contaminated or microbes. In someembodiments, the filter feature an effective pore size of 0.4 um orless, 0.2 um to 2 um, 1 um to 10 um, 5 um to 20 um, 10 um to 50 um. Itis known in the art that filters that feature an effective pore size of0.4 um or less are preferable for maintaining sterility. However, afilter such as the Porex 4901 possess a 25 um effective pore size hasbeen shown to be effective in maintaining sterility.

In some embodiments, the pressure lid comprises one or more gaskets. Insome embodiments, the one or more gaskets are adapted to permit orimprove a pressure seal (which may nevertheless not be a perfect seal).In some embodiments, at least one gasket is disposed on areservoir-contact surface of the said lid. In some embodiments, at leastone gasket is disposed on an instrument-contact surface of the said lid.In some embodiments, a gasket is adapted to permit or improve pressureseals with a plurality of reservoirs. In some embodiments, a gasket isadapted to permit or improve pressure seals at a plurality ofinstrument-facing ports. In some embodiments, the one or more of thegaskets comprise an elastomer, pliable material, O-Ring and/or acombination thereof. In some embodiments, one or more of the gaskets areformed by extrusion, casting, injection molding (includingreaction-injection molding), dye cutting and/or a combination thereof.In some embodiments, at least one gasket is mechanically coupled to thelid by adhesion (e.g. using adhesive tape), clamping, screwing down,bonding, heat-staking, welding (e.g. ultrasonically, by laser), fusing(e.g. using solvent-assisted bonding), and/or a combination thereof.

For example, one of our present embodiments of the lid includes a port(5) that allows pneumatic (e.g. vacuum) control of (optional) chipstretching to be communicated through the lid (see FIGS. 2A-2E). It isnot intended that the lid be limited to communicating only pneumaticpressure; it is contemplated that the lid can communicate additionallyfluidic or electrical interfaces.

In one embodiment, the lid can include sensors. For example, the lid maycomprise a pressure sensor to determine, for example, the pressureincident on one or more reservoirs. Further, the lid may includeliquid-level sensing to determine the amount of liquid present in thereservoir or whether specific fill (or depletion) thresholds have beenpassed. This can be done in a variety of ways. In one embodiment, thedetecting liquid optically using the difference of refractive indexes iscontemplated. In this embodiment, air-filled compartments and channelsdisperse light, while liquid or fluid-filled channels focus light. Morespecifically, the refractive indexes of liquid are from 1.3 to 1.5 whilethat of air is only 1.0. In one embodiment, each optical sensor consistsof a matched pair of an IR emitter (SEP8736, 880 nm, Honeywell) and aphototransistor (SDP8436, 880 nm, Honeywell). In this embodiment, IR ischosen over visible light for it is less susceptible to interferinglight.

The ability to easily remove fluids from the various reservoirs (e.g.take sample, replenish media, add test agents, etc.) is a desiredfeature. An especially desired feature is to be able to use standardlaboratory pipettes and syringes for such operations. However, suchfluidic access (especially using a pipette) requires the accessedreservoir to be open to the environment. This, in terms, is undesirableparticularly when the chip or disposable are in transit or in useoutside of the instrument, as the opening can provide a means forcontamination of the reservoir. A typical solution to this problem is toinclude a lid that can be applied to one or more of the reservoirs whenthey are not being accessed. However, including a simple lid cancomplicate the use of the technology, since the user typically wouldhave to actively install and remove the lid, as well as maintain lidsnear the instrument in a sterile way.

One solution is to include a means for automatically removing and/orinstalling lids as part of the system (whether integrated in the cultureinstrument or a separate module). For example, the system can include amechanical actuator that is capable of engaging a lid installed on adisposed perfusion disposable and removing it prior to engagement withthe pressure system. This mechanical actuator can re-install the lidupon removal of the perfusion disposable.

In an alternate embodiment, the system includes a means for applying alid to a perfusion disposable prior to or upon removal, for example,with the lid originating from a magazine of stored lids.

A shortcoming of the system with the means for automatically removingand/or installing lids (discussed in the prior paragraph) is that itrequires one or more mechanical actuators whose operation can bechallenging in practice. Another challenge is the following: the designof the reservoirs and in particular its opening aims to satisfy thedemands of liquid access (e.g. manual sample taking or replenishingusing a pipette), the pressure-driven system (e.g. ensuring a goodpressure seal against the instrument) and manufacturing (e.g.injection-molding of the reservoirs). In practice, these requirementscan oppose each other. For example, manual access may demand a broadreservoir opening; in contrast, it may be desirable for the pressureinterface to be narrower, to reduce the force on the instrument.

A better solution disclosed herein is to include a “pressure lid” (seeFIGS. 2A, 2B, 2C and 2D). This pressure lid is a lid that may beinstalled on to the reservoirs to reduce the likelihood ofcontamination, and is designed to stay predominantly in place while theperfusion disposable is engaged with the instrument. In order to staypredominantly in place while engaged with the instrument, the lidpreferably includes a) one or more features designed to interface withthe instrument (e.g. to received positive or negative pressure), b) oneor more features designed to interface with one or more reservoirs (e.g.create a pressure seal or minimize gas leakage so that pressure can beapplied to the reservoir), and c) a means for pressure to becommunicated from at least some of the features (a) and at least some ofthe features (b). The pressure lid or portions thereof may betransparent or translucent. This can allow, for example, viewing liquidlevels within the reservoirs. The pressure lid may include markings thatindicate the nature or name of respective reservoirs.

In one embodiment of the pressure lid, the opening in the pressure lid(e.g. on its top) may be smaller than the reservoir, to reduce thesurface area open for contamination and/or reduce the area subject to apressure seal. In another embodiment, the lid may include a filter or aplurality of filters (38) to prevent solids and particles from entering(see FIG. 2A). For example, the lid may include a 0.2 um or 0.4 umfilter known to reduce entry of bacteria and other contaminants. Manymaterials and technologies can be used for such filters. For example,track-etched filters (e.g. PTFE, polycarbonate, PET), paper filters,porous and expanded materials (e.g. cellulose and derivatives,polypropylene, etc.), sintered materials (e.g. Porex filters) may beused since the filter need only conduct pressure and not liquids.

In one embodiment, the lid may include a means for permitting gas flowbut predominantly no liquid flow. This can include, for example,hydrophobic porous membranes or filters, gas permeable membranes orfilters, etc. This approach can also help reduce the likelihood ofspillage.

In one embodiment, the lid may include a deformable portion that candeform to conduct pressure. For example, this can be an elastic orplastic membrane that stretches into the reservoir as positive pressureis applied. Similarly, the lid may include a plunger used to transmitpressure from the instrument to one or more reservoirs. Care must betaken to ensure that the desired pressure is applied to the inside ofthe reservoir, as the membrane or plunger can apply a back force. Thiscan be done, for example, by a) ensuring that the back force is small orunderstood through design of the membrane, plunger or the operatingpressure range, b) measuring the pressure inside the reservoir and usingit to control the applied pressure, c) monitoring the resulting flow tocontrol the applied pressure. The deformable portion offers one way forpressure to be communicated.

Either side of the pressure lid (instrument-facing or perfusiondisposable-facing) as well as each of the opposing surfaces (instrumentand perfusion-disposable features that interact with the pressure lid)can be designed to enable a pressure seal in a number of different ways.In one embodiment, the present invention contemplates one or moreregions comprising one or more elastic or pliable materials. In oneembodiment, this is done with one or more gaskets (see FIG. 2A), whichcan be made for example from elastomeric or pliable materials (e.g.silicone, SEBS, polypropylene, Viton, rubber, etc.). The gaskets can beshaped in a variety of ways, including cut flat sheets, o-rings (notnecessarily round in shape or cross-section), etc. In one embodiment,this is done with one or more ridges that act as load concentrators (seeFIG. 2C). Without wishing to be bound by theory, these act to localizethe sealing force to create elevated localized sealing pressure. Theseridges may potentially engage a gasket or pliable material on theopposing surface. Care must be taken to design the shape of the ridge(particularly the portion of the shape that engages the opposingsurface), as this shape can have a substantial effect on the requiredsealing pressure. A variety of shapes are contemplated (e.g.rectangular, triangular, trapezoidal, half-circular or circular section,etc.). In one embodiment, the sealing tooth has a trapezoidal shape forimproved sealing (see FIG. 2C). Alternatively, the gasket could beintegrated into either the Reservoir or Lid in the form of an overmoldedelastomer (e.g. silicone, SEBS, etc). This overmolded elastomer couldthen, itself, have an appropriate shape to act as a seal (e.g. a toothor o-ring half-round section).

The approach need not be limited to a single design. In one embodiment,the present invention contemplates a combination of one or more regionscomprising one or more elastic or pliable materials. Moreover, gasketingor ridges can be done per-reservoir, so that each is isolated in termsof applied pressure, or it can encompass two or more reservoirs, whichmay reduce complexity. In one embodiment (see FIG. 2D) the pathencircles all chambers of the reservoir chamber—cover assembly seal, soeach chamber is isolated from the other. In one embodiment (see FIG.2D), there are two reservoirs, each with an inlet chamber (6A, 6B) andan outlet chamber (7A, 7B), and a separate (optional) vacuum chamber (8)that allows for transfer of a vacuum to the chip or other microfluidicdevice. In one embodiment (FIG. 2E), the reservoir chamber—coverassembly seal comprises a sealing tooth (9).

It is not intended that the present invention be limited to a designthat has a perfect pressure seal, as this may not be required. Rather,some amount of gas leakage can be tolerated, since the instrument mayactively regulate pressure, thereby compensating for the leak. Therelaxing of a requirement to obtain a perfect seal on one or both sidescan simplify design and reduce costs.

The pressure lid can be affixed or rest upon the reservoirs (whether onthe perfusion disposable or directly on chip) in a variety of differentways. Embodiments can involve instances wherein the liquid or gas sealbetween the lid and reservoir(s) is present even outside of theinstrument (e.g. the lid is held tightly in place by something otherthan the instrument), and wherein the seal is created by action of theinstrument (e.g. the instrument presses the lid against the reservoirsduring perfusion). In another embodiment, the present inventioncontemplates a combined approach, e.g. the lid is designed to create atleast a partial seal as in the first option above, but the seal isapproved or assured by action of the instrument as in the second optionabove. An advantage of approaches that provide at least some degree ofsealing of the lid against the reservoir even outside of the instrumentis that they may reduce the risk of spills and contamination (e.g. dueto handling or transport).

Examples of approaches to affix or rest the pressure lid (regardless ofwhich of the above three approaches they fall under) include a) wherethe lid can simply rest upon the reservoirs or perfusion disposable(this can be aided by overhanding portions of the lid, so that the lidcannot simply slide off); b) the lid can be screwed, glued or pinnedinto place; and c) the lid can be clipped into place. In an alternativeembodiment, it could also be held down by a spring, e.g. a hinged lidwith a spring that forces the lid closed.

Clip features may reside in the lid, the perfusion disposable, chip orcombination thereof. Furthermore, some embodiments make use of aseparate substrate that provides clipping elements (i.e. a separatepiece that one brings in to clip the lid into place). An advantage ofthe clipping approach is that it can facilitate easy application andremoval of a lid while still securing the lid in place. The clipping maybe optional; for example, it may be applied when shipping ortransporting the device and ignored during regular use.

In some embodiments, the lid is asymmetric or includes lock-and-keyfeatures to ensure that the lid is correctly oriented with respect to aperfusion disposable and/or an instrument.

Many of the features of the perfusion disposable (PD) could potentiallybe included in the “chip” itself or a different device for coupling to achip. If the reservoirs, for example, are included in the chip, onecould use a pressure lid directly on top of the chip.

While the pressure lid has been discussed above in connection with thepressurization of one or more reservoirs within a perfusion disposableor perfusion manifold assembly, it is not intended that the pressure lidbe limited by use with only these embodiments. Indeed, it iscontemplated that the pressure lid can be used with other microfluidicdevices. The pressure lid can be movable or removably attached to othermicrofluidic devices to allow improved access to elements (e.g.reservoirs) within. The pressure lid can be removed from such otherdevices, and the other devices can be used without the lid. In oneembodiment, the other microfluidic devices comprise cells on a membraneand/or in or on one or more microchannels.

B. Tray System

It is desirable to be able to remove chips and/or perfusion disposablesfrom the instrument without having the remove the instrument itselffrom, for example, an incubating enclosure. It is also desirable to beable to remove groups of chips and/or perfusion disposables together.This is because the operations that are performed on thechips/disposables often need to be done in batches at a time (e.g. mediareplenishing, dosing with an agent, sample taking), regardless ofwhether the operations are performed automatically or manually. Forexample, it is convenient to remove groups of chips/disposables at atime if only to help transport them to a bio-safety cabinet or culturehood.

To address these needs, the present invention contemplates, in oneembodiment, a system in which perfusion disposables can be inserted orremoved from an instrument (or module) in groups by means of a traysystem (see FIG. 6). For example, a current embodiment allows eachinstrument to accept two trays (or racks) of six perfusion-disposableseach (8A and 8B).

In one embodiment, the tray (or rack) (32) may facilitate the alignmentof the perfusion disposables (10) with the instrument (30) (e.g.aligning reservoirs or port locations with corresponding pressure orfluid interfaces included in the instrument). This can be done in anumber of ways, including providing locating features for the perfusiondisposables (or any additional elements that carry them) within thetray, and providing locating features for the tray within the instrumentand alignment features (57) for the perfusion disposables (see FIG.10B). Features that can be used to support such alignment includereference surfaces, pins, guides, shaped surfaces (e.g. fillets and/orchamfers), spring or elastic elements to promote registration, etc.These may be included in the tray, instrument, perfusions disposables orcombinations thereof.

The tray may optionally be designed to capture leaks originating fromthe perfusion disposables or instrument interfaces. The tray mayoptionally include one or more optical windows that may facilitatemicroscopy or inspection. This can enable placing a tray onto amicroscope or other inspection device, allow the chips to be observedwithout having to remove each disposable from the tray. Correspondingly,the tray may be optionally designed to minimize imaging workingdistance, e.g. lay flat on or fit into a microscope stage, etc. Thesystem may optionally include a means for retaining one or more of theperfusion disposable within the tray. For example, the perfusiondisposable may clip into the tray, with clip features present on theperfusion disposable, tray, an additional substrate or combinationsthereof.

In some embodiments, the tray system includes one or more sub-trays (ornests) (47) that fit into a carrier tray (32) (see FIG. 8A). Sub-traysallow subsets of perfusion disposables (e.g. three) to be removed fromthe tray simultaneously. This can be useful, for example, where one ormore operations performed on the chips/disposables benefits from asmaller number of chips that are present on the carrier tray. Forexample, in some instances, we prefer to place no more than threedisposable on a microscope stage at one time, to minimize the time thatthe chips/disposables spend outside of their preferred incubation andperfusion environments. Consequently, a current embodiment includescarrier trays (32) that support two sub-trays (47) each, each sub-traysupporting three perfusion disposables (10) (see FIG. 8A).

The sub-trays may facilitate the alignment of the perfusion disposableswith the instrument. This can be done in a number of ways, including byproviding locating features for the perfusion disposables within thetray, by providing locating features for the sub-tray within the carriertray, and by providing locating features for the carrier tray within theinstrument. Features that can be used to support such alignment includereference surfaces, pins, guides, shaped surfaces (e.g. fillets and/orchamfers), and spring or elastic elements to promote registration, etc.These may be included in the carrier tray, sub-tray, instrument,perfusions disposables or combinations thereof. By way of an example,the present invention contemplates an embodiment wherein the perfusiondisposables align to the sub-tray, which in turn aligns to the carriertray, which in turn aligns to the instrument (see FIGS. 9A and 9B). Itis not intended that all of these alignments or necessary; indeed, somesteps in this chain may be skipped. For example, the sub-tray may aligndirectly to the instrument using any of the described features, and notrequiring the carrier tray for alignment purposes.

The sub-tray may optionally be designed to capture leaks originatingfrom the perfusion disposables or instrument interfaces. The sub-traymay optionally include one or more optical windows that may facilitatemicroscopy or inspection. This can enable placing a sub-tray onto amicroscope or other inspection device, allow the chips to be observedwithout having to remove each disposable from the tray. Correspondingly,the sub-tray may be optionally designed to minimize imaging workingdistance, e.g. lay flat on or fit into a microscope stage, etc. Thesystem may optionally include a means for retaining one or more of theperfusion disposables within the sub-tray. For example, the perfusiondisposable may clip into the sub-tray, with clip features present on theperfusion disposable, sub-tray, an additional substrate or combinationsthereof. The system may optionally include a means for retaining thesub-tray within the carrier tray. For example, the sub-tray may clipinto the carrier tray, with clip features present on the sub-tray,carrier tray, an additional substrate, or combinations thereof.

It may be convenient to divide some of the desired features between thecarrier tray and the one or more sub-trays. For example, the sub-trayscan provide an optical window and the carrier tray can be designed tocapture leaks. As this example illustrates, it may be desired to includea sub-tray even if the carrier tray is designed to support only onesub-tray.

The same instrument may support different tray or sub-tray types, aswell as different numbers of trays. For example, an instrument mayaccept two different tray types, each tray type designed for a differenttype of perfusion disposable. In such a case, the tray can in essenceact as an adaptor that adapts the different perfusion-disposable typesto the same instrument.

The present invention also contemplates in one embodiment, microscopestages, stage-inserts or adapters (e.g. that plug into the stageinserts) designed to accept one or more chips, perfusion disposables,trays or sub-trays. These can make it easy to “drop in” a number ofchips for imaging, with the chips securely retained on the stage(thereby avoiding drift, for example, as the microscope stage moves).

C. Engaging Perfusion Disposables with the Instrument

In one embodiment, the present invention contemplates a pressure-drivensystem for the biological culture in fluidic devices, which appliespressure (whether positive or negative) to one or more fluidic elements.These fluidic elements can include, for example, chips, reservoirs,perfusion disposables, pressure lids or combinations thereof. In suchsystem, the instrument interfaces with the respective fluidic element orelements in order to apply the pressure where desired. Such interfacingtypically involves establishing a gas seal, although in some embodimentsa tight seal is not required (e.g. the pressure-regulation can maintainthe desired pressure despite gas leak). Without loss of generality, thefollowing description refers to establishing a seal, but the intent isto also encompass embodiments that do not require a seal.

In the present disclosure, a system and method are contemplated forestablishing a pressure interface between a biological cultureinstrument and one or more fluidic elements. In particular, a system iscontemplated wherein, in one embodiment, the one or more fluidicelements are lifted into contact with one or more pressure manifoldsincluded in the instrument, the said one or more pressure manifolds arelowered into contact with the said one or more fluidic elements, or acombination thereof. In some embodiments, the said raising or loweringengages multiple fluidic elements with the instrument in unison (e.g.through a single operation or single movement) (see FIGS. 9A and 9B),simultaneously linking a plurality of microfluidic devices (such as oneor more of the embodiments of the perfusion manifold assembly discussedherein).

Some embodiments wherein the fluidic elements are raised include one ormore platforms onto which one or more of the fluidic elements aredisposed. In such embodiments, one or more of the platforms may beraised in order to affect the said raising of the one or more fluidicelements (FIG. 6). In some embodiments, the instrument or systemincludes a mechanical means (35) for manually achieving the said raisingor lowering involved in the said establishing of a pressure interface.Such mechanical means (35) for manual actuation can include the movingof a user-accessible control surface, which may include, for example, alevel, pull/push knob, rotational control, or combinations thereof.

In some embodiments, the instrument or system includes a mechanicalactuator (51) in order to facilitate the raising or lowering involved inthe said establishing of a pressure interface (See FIGS. 9A and 9B).Such mechanical actuator can involve, for example, one or more pneumaticcomponents (52) (e.g. cylinders), hydraulic components (e.g. cylinders),solenoids, electrical motors, magnets (e.g. fixed magnets mechanicallymoved into place), or combinations thereof. In some embodiments, themechanical actuation can be under computer control. In some embodiments,the mechanical actuation is augmented with manual control (e.g. usingany of the means for mechanical control described above), for example,in order to provide a manual override. A user interface on theinstrument can control this process.

Regardless of whether the actuation is manual or automatic, the systemcan, in some embodiments, further include one or more mechanisms forincreasing the applied mechanical force. This may be desirable in orderto provide sufficient force on the pressure interface in order to obtaina sufficient or sufficiently robust seal. Such mechanisms for increasingthe applied mechanical force can include levers, cams, pneumatic orhydraulic amplifiers, or combinations thereof.

In some embodiments, the mechanical motion can be controlled and orconstrained using various mechanical components or designs known in theart. These mechanical components or designs include, for example, rails,guide rots, pivots, cams, four-bar linkages, etc. It is important tonote that the raising or lowering motion can, but need not, be linear.For example, a rotational motion (e.g. in the case of a pivot) or acompound motion (e.g. in the case of a linkage) are desirable in someembodiments.

Although the forgoing describes raising or lowering and features presenton the top of bottom of various substrates, one with typical skill inthe art would appreciate that the description can also be applied tolateral motions or motions along other axes (and not necessarily linearmotions), and to features present on any sides or orientations.Additionally, although the forgoing implies that the one or more fluidicelements are disposed beneath the one or more pressure manifolds, onewith typical skill in the art would appreciate that the said pressuremanifolds may instead lie beneath the said fluidic elements (forexample, the pressure interfaces may be disposed on the bottom surfaceof a perfusion disposable).

A current embodiment (illustrated in the attached figures) includes twomechanics, each of which permits 6 perfusion disposables to beinterfaced with a pressure manifold (50) in a single motion. In thisembodiment, the pressure manifolds are lowered (FIG. 9B) into contactwith the perfusion disposables (or optionally in contact with pressurelids covering the perfusion disposables) using an electricallycontrolled pneumatic actuator. The force of the actuator is directedusing a cam system, which also increases the applied force due to itsmechanical advantage. The illustrated mechanism is also bi-stable, i.e.once the actuator pushes the manifold up or down it can be unpowered,while maintaining the position of the manifold. This can help with heatreduction.

D. Pressure Manifolds and Distribution Manifolds

In many applications of the pressure-driven system, it is desirable todistribute one or more pressure sources to two or more fluidic elements(including, for example, fluidic chips, perfusion disposables,reservoirs, pressure lids, or combinations thereof). For example, it maybe desirable for two or more perfusion disposables to share a single setof pressure regulators in order to reduce the number of regulators inthe system (e.g. in contrast with providing a different set ofregulators for each perfusion disposable).

In one aspect of the present disclosure, the instrument includes one ormore distribution manifolds. The said distribution manifolds includesone or more fluidic conduit (e.g. gas channels or tubes) adapted todistribute one or more pressure sources to two or more fluidic elements(e.g fluidic chips, perfusion disposables, reservoirs, pressure lids, orcombination thereof). Correspondingly, the distribution manifold mayinclude one or more pressure input ports, which may for example beadapted to communicate with one or more pressure regulators (each inputport may communicate with a single or multiple regulators). Thedistribution manifold, in one embodiment, can also have pressureregulation components (valves, pressure sensors, pressure source)integrated into the manifold itself. Similarly, the distributionmanifold may include two or more interfaces, which may for example beadapted to communicate with one or more fluidic elements. In someembodiments, the two or more interfaces include at least one regioncomprising an elastomeric or pliable material. Examples include gaskets,o-rings, etc. made of materials including silicone, SEBS, polypropylene,rubber, Viton, etc. Such regions comprising an elastomeric or pliableregion can aid in providing or improving a fluidic seal. Suchelastomeric or pliable regions can also be included in pressuremanifolds that are not distribution manifolds to provide similaradvantages.

In addition to distributing pressure that can be used, for example, toproduce pressure-driven flow, the distribution manifold may distributepressure used for other purposes, for example, to produce mechanicalstrain or compression (e.g. in actuating mechanical forces inorgans-on-chips), to create gas flow within the fluidic element.Moreover, the distribution manifold may optionally distribute one ormore liquids. Such liquids can include, for example, wash solutions,disinfectant solutions, working liquids (e.g. for liquid-handling orflow control purposes), tissue-culture media, test agents or compound,biological samples (e.g. blood), or combinations thereof. In someembodiments, the distribution manifold may comprise a working fluid, amembrane and/or a plunger disposed to conduct pressure. For example, aworking fluid may be used to reduce the amount of gas required in orderto establish a desired pressure, or to facilitate more precisevolumetric control. A membrane, plunger and/or working fluid can be usedto isolate fluids used in different parts of the distribution manifold(e.g. isolate 5% CO2 tissue-culture gas on the “reservoir side” of thedistribution manifold from dry air on the actuation side).

In many applications, it is desirable to enable proper function of theinstrument even when fewer fluidic elements are engaged than theinstrument can accept. For example, it is often desirable that aninstrument that includes a distribution manifold designed to interfacewith six perfusion disposables still support proper operation of theinstrument when only four perfusion disposable are present. For example,it may be undesirable to gas to escape through the interfaces intendedfor the missing perfusion disposables, as such gas escape may reduce gaspressure or deplete gas supplies. Such considerations are relevant evenwithout a distribution manifold (i.e. with a non-distributing pressuremanifold).

According to one aspect of the present disclosure, a pressure manifold(or specifically a distribution manifold) can include one or more valvesadapted to controllably shut-off one or more of the fluidic (e.g. gas)conduits included in the manifold. A variety of valves suitable areknown in the art, including for example pinch valves, screw valve,needle valve, ball valves, spring-loaded valves, poppet valves, umbrellavalves, Belleville valves, etc. In some embodiments, one or more of thevalves are controlled by a user. For example, a user may configure thevalves to match the configuration of perfusion disposables in use. Insome embodiments, one or more of the valves are controlledelectronically. For example, software may configure the valves accordingto knowledge of experimental settings or other information available toit. In some embodiments, one or more of the valves are controlled bysensing whether the intended fluidic element is present, for example, inorder to shut off a gas line if the fluidic element is missing. Suchsensing can involve electrical means (e.g. contact switches, conductorsclosing circuits), optical means (e.g. optical gates), magnetic means(e.g. magnetic switches), or mechanical means (e.g. levers, buttons). Insome embodiments, one or more of the sensing elements affects one ormore of the valves by means of interposed software or electronichardware. In some embodiments, one or more of the sensing elementsaffects one or more of the valves directly (e.g. by mechanical couplingor by electrically signaling to the valve). As a specific example, thepresence of a perfusion disposable can act to depress a protrudingfeature, which in turn affects the state of a valve. In someembodiments, such configuration lends itself well, for example, to pinchvalves, spring valves, poppet valves, or umbrella valves, as thedepressed protruding feature can act directly on the valve to augmentflow.

In some embodiments, it is desirable or convenient to include the saidone or more valves at one or more of the interfaces to the fluidicelements. This may be desirable, for example, since a number ofsuccessful valve designs are known that respond to a force present attheir outlets. Examples of such valves include Schrader valves, Dunlopvalves, Presta valves, umbrella valves, their modifications, and relatedvalves. As a specific example, a Schrader valve may be integrated at aninterface to a pressure lid such that when the pressure lid is present,it acts to depress the central stem of the Schrader valve, therebyallowing gas flow.

Valves suitable for inclusion in the interfaces to the fluidic elementas described above often have their control feature (e.g. the pin of aSchrader valve) (FIG. 11A) located in the middle of the valve. This,however, can pose a difficulty in some potential embodiments, since acorresponding feature must be provided on the fluidic element to depresssuch a central control feature. An alternative approach is describedherein. As illustrated in FIGS. 11A and 11D, the pressure manifold (50)or distribution can manifold can include a valve (59) such as a Schradervalve (or any listed above) and further include a shuttle (61). The saidshuttle includes a first surface that faces the location of a potentialfluidic element, and a second surface that faces the said valve. Thefirst surface is designed to accept contact from the fluidic element atthe desired location. For example, the first surface can be designed toaccept contact from the periphery of a port that may be present on,e.g., a pressure lid (11) (FIG. 11D). The second surface, in term, isdesign to mechanically engage the said valve's control surface, whichmay for example lie in the center of the valve. A further advantage ofthis approach is that the thickness of the shuttle can be adjusted, forexample, to control at what distance from the fluidic element the valvewill open.

As further illustrated in the FIGS. 11A and 11C, the interface can beoptionally covered at least in part by an elastic, pliable or deformablesubstrate, such as a pliable membrane (e.g. silicone membrane) (60). Thepresence of this elastic, pliable or deformable substrate can aid in thesealing of the fluidic element against the manifold (50). The elastic,pliable or deformable substrate can, for example, be a membrane, agasket or a suitably shaped plug, and it may comprise, for example,silicone, SEBS, Viton, polypropylene, rubber, PTFE, etc. As illustrated,the elastic, pliable or deformable substrate can be held in place bycapturing it with an additional component (e.g. a cover plate (63) inthis example). However, the elastic, pliable or deformable substrate canalso be retained in a variety of other ways, including for example bybonding, adhesion, welding, etc.

The desired function of the embodiments illustrated in FIGS. 9B, 11A and11D are hereby illustrated by example: a pressure lid (11) of aperfusion manifold assembly (10) possessing a ridge around itsinstrument interface is brought into contact with the pressure manifold(50). As the lid is moved closer to the valve, the lid's ridge beginsforming a pressure seal against the manifold's silicone membrane. Withthe lid's advance, the shuttle gradually moves up and at some pointbegins depressing the central pin or poppet (65) of the Schrader valve(59). However, according to the example, the shuttle would be designedsuch that a sufficiently good gas seal is formed before the valve's pinis depressed enough to open the Schrader valve (59). Once the valve open(and ideally not before) gas is able to flow between the manifold (50)and pressure lid (11). It is important to note that in this example,Schrader valves sense the presence of each pressure-lid ridgeindependently, rather than sensing the presence of a perfusiondisposable (or pressure lid) as a single unit. Such embodiments mayprovide a further advantage in that they may accept differentconfigurations of pressure lids or perfusion disposables, for example, aconfiguration that employs only 4 of the 5 illustrated ports.

FIG. 10A illustrates one embodiment of the PD engaging face (54) of apressure manifold (50) that is a distribution manifold and showselastomeric regions, which act as gaskets to improve gas seal againstthe fluidic element. In a current embodiment, a gas seal can be foamedby compressing these elastomeric regions against ridges present on thetop of pressure lids (11), which are in turn disposed onto perfusiondisposables (10). The illustrated distribution manifold (50) candistribute to each of six pressure lids pressure (positive or negative)used for enact pressure-driven flow as well as pressure (positive ornegative) used to actuate mechanical stretch within the includedorgan-on-chip devices (in this example, each of these is disposed withina perfusion disposable, which is in turn covered with a pressure lid).The illustrated distribution manifold includes several Schrader-likevalves (see FIG. 11D).

As the manifold engages the PDs, the valve seals engage the sealingteeth or ridges on the top of the cover (see FIG. 2C) forming a seal fortransferring pressurized gas from the manifold into the reservoirchambers. The poppet (65) (FIG. 11D) acts as a backing to provide arigid surface for the sealing tooth on the cover to compress the valveseal. This provides load transfer from the cover to the Schrader valve(59) to actuate it when a PD is in position. Simultaneously, theSchrader Valve (or similar type valve system) is actuated by theengagement to the PD Cover to all gas flow from the pressure regulatorinto the PD. When no PD is in the respective position, the valveprevents any gas flow.

The spring shuttle (55) (FIG. 10B) provides the load to the coverassembly (11) to create the reservoir chamber-cover assembly seals (e.g.pressure lid-to-reservoir seals) (FIG. 2D). In operation, there is adeflection of the valve seal and the displacement of the poppet (65)when the PD is engaged.

Alternatively, a lid compressor (FIG. 10C) provides the load to thecover assembly to create the reservoir chamber-cover assembly seals(e.g. pressure lid-to-reservoir seals).

In one embodiment, each valve assembly has an optional spring, flexureor elastic component built in that allows for pressure to be applied toeach seal independently. In one embodiment, the spring (or similarelement) is an integral part of the valve function, but one can getadditional function out of it by using it to apply pressure to thesealing tooth on the reservoir lid. The spring (or similar element) canwork to restore the shuttle and to apply pressure against the fluidicelement to provide or improve the gas seal. Independently applying thisload to each sealing element on the lid results in a design that is morerobust both to variations due to manufacturing tolerances, and how manyPDs happen to be loaded into the instrument.

In some embodiments, one or more of the described valves are controlledby software or a user. For example, the user or software may aim todisconnect gas flow even if a fluidic element (e.g. perfusiondisposable) is present at the corresponding interface. This could bedesired, for example, if the user suspects or the software or sensordetects that there is excess gas flow to the fluidic element, perhapsbecause the element is damaged. The pressure manifold (whether adistribution manifold or not) may further include sensors, for example,pressure sensors, flow sensors, etc.

E. Controlling Pressure and Flow

In one embodiment, a flow rate of between 5 and 200 uL/hr, and morepreferably between 10 and 60 uL/hr, is desired through the one or moremicrochannels of the device. In one embodiment, this flow rate iscontrolled by the applied gas pressure from the pressure manifold(described above). For example, when one applies between 0.5 and 1 kPa,this nominal pressure results, in one embodiment, in a flow rate ofbetween 15 uL/hr and 30 uL/hr.

In addition to maintaining control over this gas pressure over time (andthereby maintain control over flow), in some embodiments, one must alsoaddress the gas pressure that may be applied by the process of engagingor disengaging the manifold against the perfusion disposable. That is tosay, it is been observed, in a particular embodiment, that the step ofengaging the manifold results in a pressure spike of as much as 100 kPaon the gas present within a reservoir included in the perfusiondisposable. This can cause a spike in the flow rate and/or an undesiredpressure on a coupled microfluidic device. In the particular casewherein the coupled microfluidic device comprises a membrane, anundesired pressure spike may deform the membrane, create trans-membraneflow and/or damage any included cells.

Without being bound by theory, the described pressure spikes can becaused because the mechanical force applied by the manifold to thepressure lid deforms one or more compliant materials included in thepressure lid or perfusion disposable (e.g. compressing any gaskets andthe like). Such deformation can act to shrink the volume of gas presentin the reservoir, increasing its pressure. The opposite effect leadingto a negative spike in pressure may occur during manifold disengagement;one skilled in the art will appreciate that while this discussionprimarily contemplates positive spikes that are typical to manifoldengagement, analogous consideration may be given to negative pressurespikes that may be typical during manifold disengagement. Whetherpositive or negative, spikes can be particularly troublesome where thegas volume in the reservoir is low, which may occur when the volume ofliquid in a reservoir is high (for example, in the preferred embodimentwhen more than 3 milliliters, and particularly when the volume is morethan 5 milliliters). These engagement spikes may take time to dissipate,as the excess pressure must typically vent. In embodiments wherein thepressure lid includes a filter, this filter may provide the dominantresistance to the venting, dictating the dynamics of pressure-spikedissipation. In one embodiment, the present invention contemplatesreducing the venting resistance in the system so as to avoid, reduce themagnitude and/or reduce the duration of such spikes. In on embodiment,the present invention contemplates selecting filters in order tomitigate the pressure spikes during cartridge insertion and removal.

In this regard, reference is made to FIG. 2A-2E-2. FIG. 2A is anexploded view of one embodiment of the cover assembly (11) comprising acover or lid having a plurality of ports (e.g. through-hole ports)associated with filters (38) and corresponding holes (39) in a gasket.FIG. 2B shows the same embodiment of the cover assembly with the filters(38) and gasket positioned within (and under) the cover. In oneembodiment, the filters for the outlet pressure ports are selected forlow gas-flow resistance. For example, some embodiments employ 25 micronfilters instead of 0.2 micron filters (used in the inlet pressureports), in order to decrease resistance and cause themanifold-engagement related gas pressure (discussed above) to rapidlydissipate, avoiding a prolonged spike in the flow rate. In particularembodiments, filters with an average pore size of 25 um (commerciallyavailable from Porex, filter 4901) do not compromise sterility when ⅛inch in thickness. These filters maintain sterility, despite theirlarger pore size (much larger than typical bacteria/spores), by creatinga tortuous path through their thickness, which is significantly thickerthan the previously mentioned filter membrane/sheets.

It is important to note that the design of inlet and outlet pressureports may demand different treatment with regards to the ventingresistance. For example, in embodiments wherein the perfusion disposableor microfluidic device comprise a resistor, pressure applied on theresistor side (whether the resister is placed upstream or downstream ofa region of interest) typically does not act directly on the region ofinterest (which may, for example, include cells). This can be the case,for example, if liquid flow through the resistor generates a pressuredrop. In contrast, pressure spikes on a side without the resistor(whether inlet or outlet) may act directly on the region of interest, asthere may not be a sufficient pressure drop to provide some degree ofinsulation. In a particular example with a resistor on the inlet side ofthe region of interest, a pressure spike on the inlet may produce acorresponding spike in flow rate but minimal increase in the pressureexperienced within the region of interest; in contrast, a pressure spikeon the outlet may produce both a spike in flow rate and in experiencedpressure. In some applications, for example where the microfluidicdevice includes a membrane, pressures in the regions of interest may besignificantly more detrimental than a temporary spike in flow rate.Accordingly, in this example it may be advisable to includelow-resistance filters only in the outlet ports and include more typical(higher resistance) filters in the inlet ports, as these can provideadvantages in flow regulation (discussed further in the presentdisclosure).

Having discussed the engagement/disengagement spike issue, the issue ofcontrolling gas pressure, particularly in low pressure ranges is nowaddressed. Some commercially available pressure regulators (or pressurecontrollers) advertise an addressable pressure range with a lowerpressure limit that is greater than zero. For example the SMC ITV-0011regulators are marketed for pressure control in the range of 1 to 100kPa (it has been observed that their linearity is poor in the 0 to 1 kParange). In some applications, it may be desirable to nevertheless attainflow rates that correspond to pressures below the commercially availableregulator's specified or linear range. Moreover, the accuracy ofcommercially available pressure regulators is typically a percentage of“full range,” implying that control at the low end of pressure ischaracterized by a larger percentage of variability. In someapplications this can translate into low accuracy or fidelity inpressure control towards the lower end of the usable range. In oneembodiment, either or both of these challenges are addressed by a formof “pulse width modulation” included in a method for pressure actuation.

In this regard, reference is made to FIG. 6. In one embodiment, theculture module (30) comprises a removable tray (32) for positioning theassembly-chip combinations, a pressure surface (33), and pressurecontrollers (34). In one embodiment, the tray (32) is positioned on theculture module (30) and the tray (32) is moved up via a tray mechanism(35) to engage the pressure surface (33) of the culture module, i.e. thecover or lid (11) of the perfusion manifold assembly engages thepressure surface of the culture module. Rather than having the pressurecontrollers “on” all of the time, they are switched “on” and “off” (orbetween two or more setpoints) in a pattern. Accordingly, the switchingpattern may be selected such that the average value of pressure actingliquid in one or more reservoirs corresponds to a desired value. Suchapproaches are analogous to the techniques of pulse-width modulation(PWM), pulse-density modulation (PDM), delta-sigma modulation (DSM) andsimilar techniques that are known in the field of electricalengineering. In the case of pulse-width modulation, for example, aregular switching period is selected. Within each period the pressureregular may be turned on for a set pressure for a desired duration andturned off for the remainder of the switching period. The longer theswitch is on compared to the off periods, the higher the total averagepressure supplied. The term “duty cycle” describes the proportion of“on” time to switching period; a low duty cycle corresponds to lowpressure, because the pressure is off for most of the time. Duty cycleis expressed in percent, 100% being fully “on.” By using this type of“pulse width modulation” with the pressure controllers, it has beenfound that the average gas pressure can be reliably maintained below 1kPa, using a regulator that does not offer linear control in that range.In a particular embodiment, the pressure regulator is used in itstypical “linear” mode for pressure between 1 kPa and 100 kPa, andswitched to pulse-width modulation using an “on pressure” of 2 kPa andan “off pressure” of 0 kPa for average-pressure setpoints between 0 kPaand 1 kPa. In other examples, pulse-width, pulse-density or delta-sigmamodulation may be used for controlling the average pressure between 0.3and 0.8 kPa.

Although the disclosed method can involve applying a pulsatile pressurepattern to the pressure lid, it has been empirically found that thefilters aid in smoothing the pressure incident on the liquid with thereservoir. Without being bound by theory, the degree of smoothingincreases with the resistance of the filter to gas flow and with thevolume of gas within the reservoir (which typically decreases the moreliquid is present). Similarly, analogy to electrical circuits indicatesthat smoothing increases with shorter switching periods. Accordingly,one skilled in the art may select a degree of smoothing by selecting theresistance of the gas filter, setting a lower bound on the gas volume,and selecting a switching period or modulation pattern.

It is important to ensure that the pressure regulator is able tocontrollably regulate pressure at a sufficient rate to reproduce thedesigned pressure modulation pattern. In some embodiments, 0.2 umfilters (Porex filter membrane) and a switching period of 10 secondsprovide desired smoothing. In other embodiments, 0.4 um filters may beused.

Detailed Description of the Preferred Embodiments

A. Drop-to-Drop Connections

A drop-to-drop connection scheme is contemplated as one embodiment forputting a microfluidic device in fluidic communication with anothermicrofluidic device, including but not limited to, putting amicrofluidic device in fluidic communication with the perfusion manifoldassembly. Putting devices in fluidic communication with each other canresult in the formation of bubbles (40), as shown in FIGS. 14A and 14B,where a first surface (87) comprising a first fluidic port (89) isaligned with a second surface (88) and a second fluidic port (90). Inone embodiment, a drop-to-drop connection is used to reduce the chanceof bubbles becoming trapped during connection. Air bubbles areparticularly challenging in microfluidic geometries because they getpinned to surfaces and are hard to flush away with just fluid flow. Theypose additional challenges in cell culture devices because they candamage cells through various means.

In one embodiment, droplets are formed on the surfaces of the devices inthe areas around and on top of the fluidic vias or ports as shown inFIGS. 15A, 16A, 16B, 16D and 17-21. When the surfaces come near eachother during a connection, the droplet surfaces join without introducingany air bubbles. In practice, maintaining alignment and stability of thedroplets during manual device manipulation is challenging. Additionally,in situations where the Bond number is high liquid tends to drain fromdevices quickly and in an unstable manner. A number of solutions areherein described to address the problems of both maintaining a stabledroplet on a device surface and guiding the drop-to-drop engagement oftwo primed devices in a controlled and robust manner.

FIG. 16A shows one embodiment for bringing a microfluidic device intocontact with a fluid source or another microfluidic device, wherein themicrofluidic device approaches from the side so as to engage a sidetrack with a portion configured to fit into said side track. FIG. 16Bshows one embodiment for bringing a microfluidic device into contactwith a fluid source or another microfluidic device, wherein themicrofluidic device approaches from the side and underneath so as toengage a side track with a portion configured to fit into said sidetrack, the side track comprising an initial linear portion and asubsequent angled portion, resulting in both a sideways and upwardmovement of the microfluidic device when engaging and traversing theside track, so as to cause a drop-to-drop connection establishingfluidic communication (FIG. 16C). FIG. 16D shows yet another approachfor bringing a microfluidic device into contact with a fluid source oranother microfluidic device, wherein the microfluidic device pivots on ahinge, joint, socket or other pivot point on the fluid source or othermicrofluidic device (with an arrow showing the general direction ofmovement).

FIG. 17 is a schematic showing a confined droplet (22) on the surface(21) of a microfluidic device (16) in the via or port, wherein thedroplet covers the mouth of the port and protrudes above the port, andwhere the port is in fluidic communication with a microchannel.

FIG. 18 is a schematic showing a confined droplet (22) above the surface(21) of a microfluidic device (16) in the area of the via or port,wherein the droplet sits on a molded-in pedestal or mount (42) andcovers the mouth of the port and protrudes above the port, and where theport is in fluidic communication with a microchannel.

FIG. 19 is a schematic showing a confined droplet (22) above the surface(21) of a microfluidic device (16) in the area of the via or port,wherein the droplet sits on a gasket (43), covers the mouth of the port,and protrudes above the port, and where the port is in fluidiccommunication with a microchannel.

FIG. 20 is a schematic showing a confined droplet (22), a portion of thedroplet positioned below the surface (21) of a microfluidic device (16)in the area of the via or port, wherein the droplet sits on a molded-indepression or recess (44) and covers the mouth of the port, with aportion protruding above the surface, and where the port is in fluidiccommunication with a microchannel.

FIG. 21 is a schematic showing a confined droplet (22), a portion of thedroplet positioned below the surface (21) of a microfluidic device (16)in the area of the via or port, wherein the droplet sits in asurrounding gasket and covers the mouth of the port, with a portionprotruding above the gasket.

FIG. 22A-22B is a schematic showing a surface modification embodimentemploying stickers for confining droplets on the surface of amicrofluidic device (16) at a port, and where the port is in fluidiccommunication with a microchannel. FIG. 22A employs a hydrophilicadhesive layer or sticker (45) upon which the droplet (22) spreads outto the edges of the sticker, constrained by a surrounding hydrophobicsurface. FIG. 22B shows a droplet (22) spreading out on a hydrophilicsurface of the device, constrained by a surrounding hydrophobic surface(45) created by one or more adhesive layers or stickers on each side ofthe port, and where the port is in fluidic communication with amicrochannel.

FIG. 23 is a schematic showing a surface modification embodimentemploying surface treatment (e.g. chemical vapor deposition, plasmaoxidation, Corona, etc.—indicated by downward projecting arrows) inconjunction with a mask (41); in one embodiment, the microfluidic device(16) is made of a naturally hydrophobic material which becomeshydrophilic upon such surface treatment where there is no mask, butremains hydrophobic where there is a mask. After the surface treatment,the mask can be removed and the channel can be filled with fluid so asto generate a droplet protruding above the surface, but constrained bythe regions that remained hydrophobic (see FIG. 17).

FIG. 24A-24D is a schematic of one embodiment of a drop-to-dropconnection scheme whereby a combination of geometric shapes and surfacetreatments are used to control the droplet. FIG. 24A shows an embodimentof the microfluidic device or “chip” comprising a fluid channel andports, having an elevated region at each port (e.g. a pedestal orgasket). When other portions of the device (i.e. portions other than thepedestal or gasket) are treated (e.g. plasma treatment) to make themhydrophilic, the naturally hydrophobic pedestal or gasket can beprotected with a mask (shown in FIG. 24A on top of the pedestal orgasket as element 41) during plasma treatment to keep it from becominghydrophilic. After plasma treatment, the mask is removed (e.g. peeledoff the surface of the pedestal or gasket). FIG. 24B shows thehydrophilic channel filled with fluid where the droplet radius isbalanced at each end (i.e. at the port openings); the droplet (22) isconstrained by the hydrophobic gasket surface. FIG. 24C shows oneportion of the microfluidic device of FIG. 24B with an upward projectingdroplet (22) approaching (but not yet in contact with) one portion ofthe mating surface of the perfusion manifold assembly, which also has aprojecting droplet (in this case, the droplet (23) is projectingdownward). FIG. 24D shows the same portion of the microfluidic device ofFIG. 24C with the upward projecting droplet (22) of the microfluidicdevice making contact with (and merging with) the downwardly projectingdroplet (23) of the perfusion manifold assembly. The droplets coalescein a controlled manner when they are on hydrophilic surfaces butconstrained by hydrophobic surfaces. As noted previously, embodimentswhere the microfluidic device approaches from above (with a downwardlyprojecting droplet) the perfusion manifold assembly (with an upwardlyprojecting droplet) are also contemplated.

FIG. 25 shows an embodiment of drop-to-drop connecting using surfacetreatments alone (i.e. without geometric shapes such as pedestals orgaskets). FIG. 25A shows an embodiment of the perfusion manifoldassembly comprising a fluid channel and a port. When other portions ofthe naturally hydrophobic mating surface (i.e. portions other than theregion around the port) are treated (e.g. plasma treatment) to make themhydrophilic, the region around the port protected with a mask (shown inFIG. 25A as element 41 covering the port and a small region of themating surface around the port) during plasma treatment to keep it frombecoming hydrophilic. After plasma treatment, the mask is removed (e.g.peeled off the mating surface around the port). FIG. 25B shows thehydrophilic channel filled with fluid to a level (e.g. height of thecolumn of fluid). In some embodiments, the formed droplet is able toresist the pressure (gravitational head) exerted by the fluid volume.This is advantageous, as it can enable drop-to-drop connection whileminimizing the dripping of the top droplet and stabilizing its size.Without being bound by theory, the drop resists the exerted pressure ofthe fluid volume because that pressure is balanced out by the surfacetension of the droplet; this surface tension is determined in part bythe droplet radius, which in turn can be controlled using designs andmethods disclosed herein; for example, when the droplet is constrainedby the hydrophobic region around the port, the radius of its surface issimilarly constrained.

FIG. 26 is a chart showing (without being bound by theory) therelationship between the port diameter (in millimeters) and the maximumhydrostatic head (in millimeters) that the stabilized droplet cansupport, assuming that the fluid has the same surface tension as water(the model does not include the reservoir meniscus). This shows that onecan work with a variety of port diameters, selecting those that cansupport substantial volumes of the water column in the channel (and ingeneral support substantial back pressures), thereby providing asignificant process window and tolerance for user manipulation. In yetanother embodiment, by adjusting the pressure on the fluid, a projectingor protruding droplet of a desired size is achieved.

It is not intended that the present invention be limited to a particularmethod for controlling the droplet size, orientation, or direction. Inone embodiment, the present invention contemplates using (or making)engineered surfaces to form stable drops. Such surfaces can beinherently hydrophilic or hydrophobic, or can be treated to behydrophilic or hydrophobic. It is not intended that the presentinvention be limited to any one technique. However, among the variousmethods of hydrophilic treatment (e.g. low-pressure oxygen plasmatreatment, corona treatment, etc.), a cleaner technology is preferred totreat Poly(dimethylsiloxane) (PDMS) microfluidic devices. In oneembodiment, the present invention contemplates using atmospheric RFplasma, so that hydrophilic surfaces can be created (on what is nomiallyhydrophobic material). See Hong et al., “Hydrophilic SurfaceModification of PDMS Using Atmospheric RF Plasma,” Journal of Physics:Conference Series 34 (2006) 656-661 (Institute of Physics Publishing).In one embodiment, masks (41) are used together with such plasmatreatments, as shown in FIG. 23. For example, a mask can be adhered toregions of the surface (e.g. made of PDMS or other polymer) of themicrofluidic device (16) prior to plasma treatment in order to preventsuch regions from becoming hydrophilic (and thereby controlling whatpart of the PDMS chip become hydrophilic and what portions remainhydrophobic). After plasma treatment, the mask (41) can be removed (FIG.24) (typically by simply peeling the mask off the surface). In yetanother embodiment, the present invention contemplates the use of plasmasurface treatment in a fluorinated environment to increase thehydrophobicity of the surface. See Avram et al., “Plasma SurfaceModification for Selective Hydrophobic Control,” Romanian J. InformationScience and Technology, Vol. 11, Number 4, 2008, 409-422.

Alternatively, such surfaces can have geometric features or shapes thatcause the droplet to form or behave in a desired manner. For example, amating surface might have a projection, platform or pedestal (42) with ageometry that allows for a droplet of particular dimensions, as shown inFIG. 18. A surface might also be topped with a structure surrounding theport from which the droplet projects, such as a gasket (43) or othermechanical seal, as shown in FIG. 19, which fills the space between thetwo mating surfaces (i.e. one surface from the microfluidic device andone from the perfusion assembly), to prevent leakage while undercompression.

Alternatively (FIG. 20), a portion of the droplet can be positioned in adepression or recess (44), such that a portion of the droplet is belowthe mating surface (21) of the microfluidic device, as shown in FIG. 20and FIG. 21. In still another embodiment, adhesive patches or stickers(45) can be placed on the surface to create hydrophilic or hydrophobicregions on the mating surface of the microfluidic device, as shown inFIGS. 22A and 22B.

In yet another embodiment, a combination of geometric features andsurface treatments can be applied. For example, a hydrophobic pedestalor gasket might be used (or made) to permit smaller droplet sizes. Mostelastomeric polymers used to make gaskets are hydrophobic. Such gasketsare commercially available, e.g. from Stockwell Elastomerics, Inc.(Philadelphia Pa., USA). On the other hand, M&P Sealing machineshigh-quality products made from materials such asPolytetrafluoroethylene (“PTFE”), Perfluorolkoxy (“PFA”), or fluorinatedEthylene (“FEP”), including soft hydrophobic gaskets (Orange, Tex.,USA). These are also contemplated in some embodiments. When otherportions of the device (i.e. portions other than the pedestal or gasket)are treated (e.g. plasma treatment) to make them hydrophilic, anaturally hydrophobic pedestal or gasket can be protected with a maskduring plasma treatment to keep it from becoming hydrophilic.

In one embodiment, the walls of the port (or at least a portion thereofleading up to the mating surface of the microfluidic device) arehydrophilic or made hydrophilic. In one embodiment, the walls of thecorresponding port (or at least a portion thereof leading up to themating surface of the perfusion assembly) are hydrophilic or madehydrophilic. In one embodiment, both the walls of the port of themicrofluidic device and the corresponding port of the perfusion assembly(or portions thereof) are hydrophilic or made hydrophilic.

In one embodiment, the present invention contemplates that the surfaceis designed to retain a droplet that resists the weight of liquid in thereservoir (as shown in FIG. 25A-25B). This is especially important inpractice, since it allows the droplets that go on the top device (i.e.where a first device approaches a second device from above) to be easilycreated. This embodiment allows one to simply put a measured amount ofliquid into the reservoir (e.g. 100 uL, 75 uL, 50 uL or some otheramount), leading that liquid to flow to the port, form a droplet andstop on its own. Importantly, it is not intended that this embodiment belimited to any particular amount of liquid; indeed, one does not need aprecisely measured amount of liquid. It is sufficient to aim for acertain amount, as long as that amount is below a certain threshold(where the weight of the water overwhelms the droplet's surface tensionand breaks through) in order to form a droplet by this method. It mightbe more or less convex depending on how much liquid is pushing down onit, but the spatial extent of the droplet should be the same.

It is not intended that the present invention be limited to only onemanner for drop-to-drop connecting of microfluidic devices. In oneembodiment, a first microfluidic device, such as an organ on a chipmicrofluidic device comprising cells that mimic one or more functions ofcells in an organ in the body (i.e. mimic one or more functions of cellsin an organ in the body such as cell-cell interaction, cytokineexpression, etc.), has a droplet projecting upward, while thecorresponding droplet on a second microfluidic device projects downward,as shown in FIG. 15A. In another embodiment, the first microfluidicdevice, such as an organ on a chip microfluidic device comprising cellsthat mimic cells in an organ in the body or at least one function of anorgan, has a droplet projecting downward, while the correspondingdroplet on the second microfluidic device projects upward.

Gravity alone, aside from momentum arguments, also plays a role instable droplet formation. For example, a chip that is laid flat on atable does not experience significant forces due to gravity. If thatdevice is tipped, as part of the engagement procedure for example, fluidwill flow from the higher to lower point. Therefore, orientation of thedevice might be considered another way to aide in the confinement ofdroplets, including which device has vias pointing upwards vs downwards.

An additional aspect of controlling droplet volume is the fluidicresistance of the device channels. If a device has small channels, forexample, the fluidic resistance might be high enough to maintain anearly constant droplet volume over time despite there being forcesdriving fluid flow out of the device (e.g. gravity or capillary force).This is true even in the case of high Bond number. Tuning fluidicresistance might be utilized as a singular method to “confine droplets”or in combination with other methods like controlling liquid pinninggeometry or controlling the wetting properties of the surfaces; fluidicresistance would be used to control droplet volume, while controllingthe wetting properties of the surface would help control dropletplacement.

B. Microfluidic Devices

It is not intended that the present invention be limited by the natureof the microfluidic device. However, preferred microfluidic devices aredescribed in U.S. Pat. No. 8,647,861, hereby incorporated by reference,and they are microfluidic “organ-on-chip” devices comprising livingcells in microchannels, e.g. cells on membranes in microchannels exposedto culture fluid at a flow rate. The surfaces of the microchannelsand/or the membrane can be coated with cell adhesive molecules tosupport the attachment of cells and promote their organization intotissues. Where a membrane is used, tissues can form on either the uppersurface, the lower surface or both. In one embodiment, different cellsare living on the upper and lower surfaces, thereby creating one or moretissue-tissue interfaces separated by the membrane. The membrane may beporous, flexible, elastic, or a combination thereof with pores largeenough to only permit exchange of gases and small chemicals, or largeenough to permit migration and transchannel passage of large proteins,as well as whole living cells. In one embodiment, the membrane canselectively expand and retract in response to pressure or mechanicalforces, thereby further physiologically simulating the mechanical forceof a living tissue-tissue interface.

FIG. 33A-33B shows a schematic of an illustrative microfluidic device or“organ-on-chip” device. The assembled device is schematically shown inFIG. 33A, which includes a plurality of ports. FIG. 33B shows anexploded view of the device of FIG. 33A, showing a bottom piece (97)having channels (98) in a parallel configuration, and a top piece (99)with a plurality of ports (2), with a tissue-tissue interface simulationregion comprising a membrane (101) between the top (99) and bottom (97)pieces, where cell behavior and/or passage of gases, chemicals,molecules, particulates and cells are monitored. In an embodiment, aninlet fluid port and an outlet fluid port are in communication with thefirst central microchannel such that fluid can dynamically travel fromthe inlet fluid port to the outlet fluid port via the first centralmicrochannel, independently of the second central microchannel. It isalso contemplated that the fluid passing between the inlet and outletfluid ports may be shared between the central microchannels. In eitherembodiment, characteristics of the fluid flow, such as flow rate and thelike, passing through the first central microchannel is controllableindependently of fluid flow characteristics through the second centralmicrochannel and vice versa.

FIG. 34 is a schematic showing an embodiment with two membranes (101 and102) with cells (103) inside the device in a first channel, but also incontact with fluid channels (104 and 105) with arrows showing thedirection of flow. This three channel device allows one to follow themigration or movement of cells, e.g. lymphoid cells, vascular cells,nerve cells, etc. In one embodiment, membrane 101 is coated with alymphatic endothelium on its upper surface and with stromal cells on itslower surface, and stromal cells are also coated on the upper surface ofthe second porous membrane 102 and a vascular endothelium on its bottomsurface. The movement of these vascular and stromal cells can bemonitored. Alternatively, a third type of cell can be placed in themiddle (103) and the migration through the membranes can be monitored(e.g. by imaging or by detection of cells in the channels or channelfluid). The membranes may be porous or have grooves to allow cells topass through the membranes.

In one embodiment this three channel device is used to determine cellbehavior of cancer cells. Tumor cells are placed, for example, in thecentral microchannel surrounded on top and bottom by layers of stromalcells on the surfaces of the upper and lower membranes. Fluid such ascell culture medium or blood enters the vascular channel. Fluid such ascell culture medium or lymph enters the lymphatic channel. Thisconfiguration allows researchers to mimic and study tumor growth andinvasion into blood and lymphatic vessels during cancer metastasis. Themembranes may be porous or have grooves to allow cells to pass throughthe membranes.

C. Seeding Devices with Cells

In many of the embodiments described above, the microfluidic chip orother device comprises cells. In some embodiments, cells are seededdirectly into the chip. However, in other embodiments, the chip iscontained in a carrier, which in turn is mounted on a stand tofacilitate cell seeding. FIGS. 35A-C show one embodiment of a “seedingguide” and stand. In one embodiment, the seeding guide engages thecarrier which contains the microfluidic chip, and holds the chip rightside up (e.g. for top channel seeding) and upside down (e.g. for bottomchannel seeding) in the various stages of seeding and/or coating (e.g.ECM coating), so as to improve aseptic technique. FIG. 35A shows how oneembodiment of a stand (100) is assembled, i.e. by engaging two end caps(106, 107) with side panels (108, 109). FIG. 35B shows a chip (16) and acarrier (17) engaged by the seeding guide, the seeding guide approachingthe stand (100). FIG. 35C shows six carriers (17) with chips, eachengaged with a seeding guide, each seeding guide mounted on the stand(100). The seeding guide is adapted to accept a chip carrier (e.g. in amanner similar to how the skirt engages the chip carrier); after coatingand/or seeding the same chip carrier can be (after disengaging from theseeding guide) linked to a perfusion manifold assembly. The seedingguide is designed to allow the chip to be held (whether right side up orupside down) such that its ports do not contact the tabletop or anyother surface. This is in order to avoid the contamination of the chipthrough such contact. Additionally, the seeding guide or holderfacilitates access to the chip through pipettes and/or needles and mayoptionally assist their insertion into chip ports using guide features.

In one embodiment, the present invention contemplates a method ofseeding, comprising a) providing i) a chip at least partially containedin a carrier, ii) cells, iii) a seeding guide and iv) a stand withportions configured to accept at least one seeding guide in a stablemounted position; b) engaging said seeding guide with said carrier tocreate an engaged seeding guide, c) mounting said engaged seeding guideon said stand, and d) seeding said cells into said chip (e.g. withpipette tips) while said seeding guide (along with the carrier and chip)is in a stable mounted position. In one embodiment, the microfluidicdevice or chip comprises a top channel, a bottom channel, and a membraneseparating at least a portion of said top and bottom channels. In oneembodiment, the microfluidic device or chip, after the seeding of stepc) comprises cells on the membrane and/or in (or on) one or more of thechannels (e.g. the top channel is seeded). In one embodiment of thismethod, a plurality of seeding guide are mounted on the stand,permitting a plurality of chips to be seeded with cells. The guide has anumber of functions, including a) keeping the surface of a chip sterileduring handling, b) guiding pipette tips properly into ports duringseeding, c) clearly labeling the channels of the chip (e.g.differentiating between the top and bottom channels), and d) permittingthe shipping of the chips with liquid in the channels (as well asshipping of chips with cells already seeded or functionalized with ECM).The stand also has a number of functions, including a) keeping the chiplevel to allow cells to distribute evenly across the membrane, b)allowing the guide to be flipped upside down for seeding of the bottomchannel, and c) enabling users to carry and store many seeded chips atone time. Thus, in one embodiment, after the seeding of step c), themethod continues with the steps of flipping the chip upside down andseeding the bottom channel.

EXPERIMENTAL Example 1

Conditions for bonding the capping layer (FIG. 2A-2E-2, element 13) tothe backplane (14) were examined. Extruded SEBS sheets were bonded to ahot embossed plate. The SEBS sheets were designed to act as the cappinglayer to the channels that are formed in the COP via the hot embossingprocess and as a fluid and gas gasketing to mating parts. The testingshowed that the 1 mm thick SEBS was better as a fluid seal between thereservoirs and the backplane. The hot embossed plates were fabricatedfrom Zeonor 1420R. The SEBS materials used were:

A. Thickness: 1 mm, Material: Kraton G1643, Mfg Process: extrusion

B. Thickness: 0.2mm, Material: Kraton G1643 +5% Polypropylene, MfgProcess: extrusion An oven process was used in comparison to alaminator. The laminator produced marginal to not adequate bonding.However, the oven process revealed the following:

Material Thickness 0.2 mm SEBS 1 mm SEBS Bonding Temp (C.) 80 80 BondingTime 1 hr-24 hr Clamping Pressure None 0.5 kg Applied through a siliconecoated acrylic plate Necessary for conformal lamination/good bondproduction Bond Quality 1 hr: good bond Good bond 24 hr: excellent bondAnisotropic Effects None noticeable Yes. Requires clamping pressure tobe held for ~30 min during cooling

In some embodiments, the fluidic layer is sealed with a film. This filmmay be polymeric, metallic, biological or a combination thereof (e.g. Alaminate of multiple materials). Examples of materials includepolypropylene, SEBS, COP, PET, PMMA, aluminum, etc. Specifically, thefilm may be elastomeric. The film may be affixed to the fluidic layer bymeans of an adhesive agent, thermal lamination, laser welding, clamping,and other methods known in the art. The film may further be used toaffix and potentially fluidically interconnect additional components tothe fluidic layer. For example, the film may be used to adhere one ormore reservoirs to the fluidic layer. In an example embodiment, the filmis a thermal lamination film that includes EVA or EMA. In the exampleembodiment, the film may be first laminated against the fluidic layerusing a thermal treatment and then, using a second thermal treatment,adheres one or more reservoirs to the fluidic layer. In a differentembodiment, the film includes SEBS, which is known to be bondable to avariety of materials including polystyrene, COP, polypropylene, etc.,either using a thermal treatment or with the help of one or moresolvents. In this example, the SEBS film may be laminated to a fluidiclayer (using thermal treatment or with the help of solvent) and using asecond treatment, bond one or more reservoirs to the fluidic layers.There are multiple potential advantages to using a film that iselastomeric, deformable, or pliable, or film that reflows during thebonding process. These advantages include, for example: potentiallyconforming to the fluidic layer or other bonded component (e.g.reservoirs), thereby relaxing manufacturing tolerance (e.g. on theflatness or planarity of the manufactured parts), potentiallysimplifying the required parallelism or alignment during bonding (e.g.because the said film may deform to absorb errors in parallelism), andacting as a gasket to create a fluidic seal, for example, between thefluidic backplane and reservoirs. SEBS is especially advantageous as abonding film, since it can bond under moderate temperatures (typicallyunder 100 C) while not significantly reflowing. Reflowing may beundesirable as it poses a risk of filling in and blocking fluidicchannels. By not significantly reflowing, SEBS can better maintain thedimensions and structure of fluidic channels and other features in thefluidic layer compared to materials that reflow (e.g. traditionalthermal lamination films). Film thickness can range from 10 um to 5 mmin different embodiments. The film may include various fluidic ports orchannels. The film need not be flat and can take on a variety ofthree-dimensional shapes.

Example 2

In this example, one embodiment of a protocol for chip activation isdiscussed. The example assumes that all work is done under a hood usingaseptic techniques and all working spaces are sterile (or made sterile).

Part I: Preparing The Chip

-   A. Spray the exterior of the chip package with 70% Ethanol and wipe    it prior to bring it inside hood.-   B. Open package inside hood and take chip in chip carrier out (keep    these together).-   C. Place chip in chip carrier within large sterile dish-   i. Only handle the chip carriers by their wings. Always use tweezer    to handle chip, The surface of chip is connected with cell culture    area. Avoid touching the surface of the chip with hands and keep the    chip unit flat-   D. Allow vial of Emulate Reagent 1 (ER1) powder (containing a    cross-linker) to fully equilibrate to ambient temperature before    opening to prevent condensation inside the storage container—ER1 is    moisture and light sensitive-   E. Turn the light in the biosafety hood off-   F. Reconstitute the powder with Reagent 2-   i. Add 1 ml of Emulate Reagent 2 (ER2) (containing a buffer)    directly into the ER1 storage container and invert 3 times to mix    thoroughly-   ii. Cover the ER1 solution with tin foil to prevent light    degradation-   G. Wash chip-   i. Orient the chip horizontally within the hood-   ii. Pipette up 100 ul of ER2 solution using tip-   iii. Place the pipette in a completely vertical position and insert    into the bottom channel—If it is hard to find the port, navigate    touching the surface near the port-   iv. After finding the port, inject the tip into the port (make tight    connection)-   v. Wash 100 ul of ER2 solution and keep the pipette plunger    depressed (if you see outlet fluid coming out, washing is done    successfully, if you see fluid coming out from the same port of    injection, tip is not injected properly, repeat step iv)-   vi. To take out the tip, gently press the chip body using sterile    tweezer and tip out, keep the pipette plunger depressed-   vii. Aspirate outlet flow-   viii. Repeat the same procedure for top channel washing-   ix. After washing, empty top channel first and bottom channel with    aspirator-   H. Inject ER1 Solution to both channels-   i. Pipette up 30 ul of ER1 solution using tip-   ii. Navigate the port of inlet of bottom channel using pipette tip    on top of the chip surface near the port-   iii. After finding the port, inject the tip into the port (make    tight connection)-   iv. Inject 30 ul of ER1 solution and keep the pipette plunger    depressed (if you see outlet fluid coming out, injection is done    successfully, if you see fluid coming out from the same port of    injection, tip is not injected properly, repeat step ii)-   v. To take out the tip, gently press the chip body using sterile    tweezer and tip out, keep the pipette plunger depressed-   vi. Aspirate excessive fluid from the surface of chip (avoid to    contact the port)-   vii. Repeat the same procedure for the top channel using 50 ul of    ER1 solution-   viii. Avoid introduction of bubbles. Inspect channels under    microscope to be sure no bubbles are present, if bubbles are    present, inject with ER1 solution again-   I. Place chips directly under UV lamps, ensure UV light unit is in    hood, light turns on, and adjust setting with button on back to    “constant”-   J. Treat UV light for 20 min-   K. After UV treatment, gently aspirate ER1 from channels via same    ports until channels are free of solution-   L. Wash with 100 ul of ER2 solution to both channels and then with    200 ul of dPBS

Part II: Coating

-   A. Prepare ECM as directed by manufacturer. It is recommended to    aliquot ECM and freeze if manufacturer instructed. Avoid multiple    freeze-thaw cycles-   B. Calculate total volume of ECM solution-   * Minimum volume for Channels-   i. Top: 50 ul-   ii. Bottom: 20 ul-   iii. ECM Diluent: User defined per ECM, prepare on ice.    -   **if using Matrigel, see Matrigel protocol** (make sure matrigel        protocol has “slushy ice, no touching, any warming will destroy        matrigel)-   C. Aspirate dPBS from channels-   D. Load channels with ECM solution-   i. Pipette up 30 ul of cold ECM solution using tip-   ii. Navigate the port of inlet of bottom channel using pipette tip    on top of the chip surface near the port-   iii. After finding the port, inject the tip vertically into the port    (make tight connection)-   iv. Inject 30 ul of ECM solution and keep the pipette plunger    depressed (if you see outlet fluid coming out, injection is done    successfully, if you see fluid coming out from the same port of    injection, tip is not injected properly, repeat step ii)-   v. To take out the tip, gently press the chip body using sterile    tweezer and tip out-   vi. Aspirate excessive fluid from the surface of chip (avoid to    contact the port)-   vii. Repeat the same procedure for the top channel using 50 ul of    ECM solution-   E. Incubate at 4° C. overnight or for 2 hour at 37° C.-   F. Seal the dish containing coated chips using parafilm.

Example 3

This example provides one embodiment of a protocol for seeding cellsinside the chip in the top channel (which is oriented horizontally,unless otherwise indicated). The example assumes aseptic techniques anda sterile environment.

It should be noted that, although some cells require very specificseeding conditions, in general an optimal seeding density is achievedwhen the cells are in a planar monolayer spaced closely. From thisspacing, most primary cells will attach and spread into a confluentmonolayer.

Reference is made below to “gravity washing.” This involves a) placing a(bolus) drop of media (100 uL) over a port on one side of the channel,making sure not to introduce any air bubbles within the port itself, andb) allowing this to flow through the chip, constantly aspirating mediaexcess from the outlet port.

-   A. Transfer the chips into the hood-   B. Place them inside of a sterile dish (eg 15 mm culture dish)-   C. Gently wash chips-   i. Pipette up 200 ul of cell culture medium using tip-   ii. Navigate the port of inlet of bottom channel using pipette tip    on top of the chip surface near the port-   iii. After finding the port, inject the tip vertically into the port    (make tight connection)-   iv. Wash 200 ul of medium and keep the pipette plunger depressed (if    you see outlet fluid coming out, washing is done successfully, if    you see fluid coming out from the same port of injection, tip is not    injected properly, repeat step iv)-   v. To take out the tip, gently press the chip body using sterile    tweezer and tip out, keep the pipette plunger depressed-   vi. Aspirate outlet fluid-   vii. Repeat the same procedure for top channel washing-   viii. Repeat washing step for both channels one more time-   ix. Add medium drop in inlet and outlet ports (100 ul each)-   D. Cover dish, and place to the incubator until cells are ready-   E. Prepare cell suspension and count cell number-   F. Seeding density is specific to the top and bottom channels, cell    type, and to the user's defined needs-   i. Top channel: e.g. Caco2 cells: 2.5 million cells/ml-   ii. Bottom channel: e.g. HUVEC: confluent-   G. After counting cells, adjust cell suspension to appropriate    density-   H. For top channel seeding, bring dish containing chips in the hood    and aspirate excess medium on the surface of chip (only handle the    chip carriers by their wings; keep the chip carrier flat—do not pick    it up! This will ensure an even distribution of cells across the    chip culture membrane)-   I. Agitate cell suspension gently before seeding each chip-   J. Pipette 50 uL of the cell suspension and seed into the top    channel (top channel is the lower right hand port when the chip is    in the horizontal position) (use one chip first)-   i. Place the pipette in a completely vertical position and insert    into the top channel (vertical is a gentler introduction into the    chip and ensures a more even cell distribution)-   ii. Inject 50 ul of cell suspension and keep the pipette plunger    depressed (if you see outlet fluid coming out, injection is done    successfully, if you see fluid coming out from the same port of    injection, tip is not injected properly, repeat step ii)-   iii. To take pipette tip out, gently press the chip body using    sterile tweezer except cell culture area and tip out, keep the    plunger depressed.-   iv. Immediately aspirate outlet fluid from chip surface using seeded    tip (avoid to contact the port)-   v. Use the pipette to immediately remove outflow from chip surface    using seeded tip * Remove the outflow so that both inlet and outlet    are even with surface of chip to prevent hydrostatic pressure flow-   K. Cover the dish and transfer to the microscope to check density-   L. After seeding, place the chips it in the incubator until cells    have attached-   i. Place a small reservoir (15 ml or 50 ml conical tube cap) with    PBS inside of the dish to provide humidity to cells-   ii. Range of attachment time is 1˜3 hours depends on cell type-   M. After cells have attached, gravity wash the chips with warm    medium by gently washing media through the channels.-   N. Return chips to incubator until ready to move on to next step

Example 4

This example provides one embodiment of a protocol for seeding cellsinside the chip in the bottom channel (which is oriented horizontally,unless otherwise indicated). The example assumes aseptic techniques anda sterile environment.

It should be noted that, although some cells require very specificseeding conditions, in general an optimal seeding density is achievedwhen the cells are in a planar monolayer spaced closely. From thisspacing, most primary cells will attach and spread into a confluentmonolayer.

Reference is made below to “gravity washing.” This involves a) placing a(bolus) drop of media (100 uL) over a port on one side of the channel,making sure not to introduce any air bubbles within the port itself, andb) allowing this to flow through the chip, constantly aspirating mediaexcess from the outlet port.

-   A. Bring dish containing chips in the hood and aspirate excess    medium on the surface of chip (only handle the chip carriers by    their wings; keep the chip carrier flat—do not pick it up! This will    ensure an even distribution of cells across the chip culture    membrane)-   B. Agitate cell suspension gently before seeding each chip-   C. Pipette 20 μL of the cell suspension and seed into the bottom    channel (the bottom channel is the upper right hand port when the    chip is in the horizontal position) (use one chip first)-   i. Inject 20 ul of cell suspension and keep the pipette plunger    depressed (if you see outlet fluid coming out, injection is done    successfully, if you see fluid coming out from the same port of    injection, tip is not injected properly, repeat step ii)-   ii. To take pipette tip out, gently press the chip body using    sterile tweezer except cell culture area and tip out, keep the    plunger depressed.-   iii. Immediately aspirate outlet fluid from chip surface using    seeded tip (avoid to contact the port)-   iv. Remove the outflow so that both inlet and outlet are even with    surface of chip to prevent hydrostatic pressure flow-   D. Cover the dish and transfer to the microscope to check density-   E. After seeding, flip the chip inside of dish and place the chips    it in the incubator until cells have attached underneath the    membrane-   i. Range of attachment time is 1˜3 hours depends on cell type-   ii. Place a small reservoir (15 ml or 50 ml conical tube cap) with    PBS inside of the dish to provide humidity to cells-   F. After cells have attached, flip chips back, gravity wash the    chips with warm medium by gently injecting media through the    channels.-   G. Return chips to incubator until ready to move on to next step    (cells can be cultured in the chip under static conditions until    ready to connect to the perfusion manifold for flow conditions)-   i. Aspirate old medium from the chip surface-   ii. Gravity rinse the chips with warm medium by gently injecting    media through the channels every day: 200 ul each for top and bottom    channel, drop the medium in inlet port-   iii. Place a small reservoir (15 ml or 50 ml conical tube cap) with    PBS inside of the dish to provide humidity to cells

Example 5

In this example, one embodiment of a protocol for preparing theperfusion disposable or “pod” is provided. This assumes aseptictechniques and a sterile environment.

-   A. Warm media to 37° C. ahead of time-   B. Transfer warmed media into the biohood-   C. Aliquot required amount +5% into 50 mL conical tubes-   D. Sanitize and transfer one steriflip vacuum filter into hood for    each tube of media-   i. Take steriflip out of packaging and connect to 50 mL tube of    media-   ii. Connect to vacuum inside of hood and invert-   iii. Use a timer to vacuum degas for a minimum of 15 min-   E. Prepare correct number of PODs (based on # of viable chips)-   F. Sanitize the Emulate nests and trays with ethanol and transfer    them into the hood-   G. Sanitize one packaged Pod for each of the viable Chips with    ethanol and transfer into the hood (always hold only edges of POD    with thumb and long finger; keep lid of POD on and flat using index    finger while simultaneously holding POD)-   H. Remove the reservoir lid and add media. This should create    droplets suitable for drop-to-drop engagement of the POD and the    Chip.-   i. Input Reservoir: Fill 1-3 ml (1 ml minimum)-   ii. Output Reservoir: 300 ul-   I. Transfer Seeded Chips from the incubator and bring to hood-   i. Remove the pipette tips with a gentle twisting motion and dispose    of them-   ii. Use a 200 μL pipette to add 10-50 μL of media over each port    (avoid creating a bubble inside the port). This should create    droplets suitable for drop-to-drop engagement of the POD and the    Chip.-   J. Connect Chip+Carrier to POD. This connection process should    result in drop-to-drop engagement of the POD and the Chip using the    droplets formed in Steps H and I.-   i. In one hand, hold a chip carrier with the index finger and thumb    pinching the carrier, with the thumb on the locking mechanism-   ii. With the other hand grasp the Pod with the thumb and long finger    around the reservoir and place the index finger on the top of the    lid to secure it-   iii. Orient the Pod so that you are looking “into” it, along the    tracks inside it-   iv. Continuing to pinch the carrier, align the feet of the carrier    with the tracks within the Pod-   v. Slide the chip carrier into the Pod-   vi. Use your thumb against the chip carrier to gently depress the    locking mechanism until it slides into place, capturing the chip    within the Pod-   vii. Confirm that each reservoir lid is correctly on each Pod

1-9. (canceled)
 10. A system, comprising a) device comprising anactuation assembly configured to move a pressure manifold, said pressuremanifold comprising integrated valves, said pressure manifold in contactwith b) a plurality of microfluidic devices.
 11. The system of claim 10,wherein said microfluidic devices are perfusion disposables.
 12. Thesystem of claim 10, wherein said valves comprise Schrader valves. 13.The system of claim 10, wherein each of said microfluidic devices iscovered with a cover assembly comprising a cover having a plurality ofports, and said pressure manifold comprising a mating surface withpressure points that correspond to the ports on the cover, wherein thepressure points of the mating surface of the pressure manifold are incontact with said ports of the cover assembly.
 14. The system of claim13, wherein said ports comprise through-hole ports.
 15. The system ofclaim 13, wherein said ports are associated with filters andcorresponding holes in a gasket.
 16. The system of claim 13, wherein thedevice further comprises pressure controllers.
 17. The system of claim16, wherein said pressure controllers are configured to apply pressurevia said pressure points.
 18. The system of claim 10, wherein saidactuation assembly comprises a pneumatic cylinder operably linked tosaid pressure manifold.
 19. The system of claim 13, wherein said matingsurface further comprises alignment features configured to align amicrofluidic device when said microfluidic device engages said matingsurface.
 20. The system of claim 13, wherein said device furthercomprises elastomeric membranes and said elastomeric membranes are incontact with said microfluidic devices.
 21. The system of claim 10,wherein said device is a culture module for perfusing cells.
 22. Thesystem of claim 21, wherein the culture module is configured to acceptone or more trays, each tray comprising a plurality of microfluidicdevices.
 23. The system of claim 21, wherein said culture module furthercomprises a user interface to control said culture module. 24-32.(canceled)